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Alpha-amylases

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20120270267 patent thumbnailZoom

Alpha-amylases


The present invention relates to alpha-amylase variants, polynucleotides encoding the variants and nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variant enzymes.
Related Terms: Alpha-amylase

Browse recent Novozymes A/s patents - Bagsvaerd, DK
Inventors: Carsten Andersen, Thomas Agersten Poulsen
USPTO Applicaton #: #20120270267 - Class: 435 696 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide >Blood Proteins



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The Patent Description & Claims data below is from USPTO Patent Application 20120270267, Alpha-amylases.

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US 20120270266 A1 20121025 1 21 1 6402 DNA Sus scrofa gene (1)...(6399) Factor VIII-- Full Length 1 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctc acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt ctc tgg gtc cta ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 aag aat gtc att gaa ccc aga agc ttt gcc cag aat tca aga ccc cct 2304 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 agt gcg agc caa aag caa ttc caa acc atc aca agt cca gaa gat gac 2352 Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp 770 775 780 gtg gag ctt gac ccg cag tct gga gag aga acc caa gca ctg gaa gaa 2400 Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu 785 790 795 800 cta agt gtc ccc tct ggt gat ggg tcg atg ctc ttg gga cag aat cct 2448 Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro 805 810 815 gct cca cat ggc tca tcc tca tct gat ctt caa gaa gcc agg aat gag 2496 Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu 820 825 830 gct gat gat tat tta cct gga gca aga gaa aga ggc acg gcc cca tcc 2544 Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser 835 840 845 gca gcg gca cgt ctc aga cca gag ctg cat cac agt gcc gaa aga gta 2592 Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val 850 855 860 ctt act cct gag cca gag aaa gag ttg aag aaa ctt gat tca aaa atg 2640 Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met 865 870 875 880 tct agt tca tca gac ctt cta aag act tcg cca aca att cca tca gac 2688 Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp 885 890 895 acg ttg tca gcg gag act gaa agg aca cat tcc tta ggc ccc cca cac 2736 Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His 900 905 910 ccg cag gtt aat ttc agg agt caa tta ggt gcc att gta ctt ggc aaa 2784 Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys 915 920 925 aat tca tct cac ttt att ggg gct ggt gtc cct ttg ggc tcg act gag 2832 Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr Glu 930 935 940 gag gat cat gaa agc tcc ctg gga gaa aat gta tca cca gtg gag agt 2880 Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser 945 950 955 960 gac ggg ata ttt gaa aag gaa aga gct cat gga cct gct tca ctg acc 2928 Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr 965 970 975 aaa gac gat gtt tta ttt aaa gtt aat atc tct ttg gta aag aca aac 2976 Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn 980 985 990 aag gca cga gtt tac tta aaa act aat aga aag att cac att gat gac 3024 Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp 995 1000 1005 gca gct tta tta act gag aat agg gca tct gca acg ttt atg gac aaa 3072 Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys 1010 1015 1020 aat act aca gct tcg gga tta aat cat gtg tca aat tgg ata aaa ggg 3120 Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly 1025 1030 1035 1040 ccc ctt ggc aag aac ccc cta agc tcg gag cga ggc ccc agt cca gag 3168 Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu 1045 1050 1055 ctt ctg aca tct tca gga tca gga aaa tct gtg aaa ggt cag agt tct 3216 Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser 1060 1065 1070 ggg cag ggg aga ata cgg gtg gca gtg gaa gag gaa gaa ctg agc aaa 3264 Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys 1075 1080 1085 ggc aaa gag atg atg ctt ccc aac agc gag ctc acc ttt ctc act aac 3312 Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn 1090 1095 1100 tcg gct gat gtc caa gga aac gat aca cac agt caa gga aaa aag tct 3360 Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser 1105 1110 1115 1120 cgg gaa gag atg gaa agg aga gaa aaa tta gtc caa gaa aaa gtc gac 3408 Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp 1125 1130 1135 ttg cct cag gtg tat aca gcg act gga act aag aat ttc ctg aga aac 3456 Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn 1140 1145 1150 att ttt cac caa agc act gag ccc agt gta gaa ggg ttt gat ggg ggg 3504 Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly 1155 1160 1165 tca cat gcg ccg gtg cct caa gac agc agg tca tta aat gat tcg gca 3552 Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Ser Ala 1170 1175 1180 gag aga gca gag act cac ata gcc cat ttc tca gca att agg gaa gag 3600 Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu 1185 1190 1195 1200 gca ccc ttg gaa gcc ccg gga aat cga aca ggt cca ggt ccg agg agt 3648 Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser 1205 1210 1215 gcg gtt ccc cgc cgc gtt aag cag agc ttg aaa cag atc aga ctc ccg 3696 Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro 1220 1225 1230 cta gaa gaa ata aag cct gaa agg ggg gtg gtt ctg aat gcc acc tca 3744 Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser 1235 1240 1245 acc cgg tgg tct gaa agc agt cct atc tta caa gga gcc aaa aga aat 3792 Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn 1250 1255 1260 aac ctt tct tta cct ttc ctg acc ttg gaa atg gcc gga ggt caa gga 3840 Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly 1265 1270 1275 1280 aag atc agc gcc ctg ggg aaa agt gcc gca ggc ccg ctg gcg tcc ggg 3888 Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser Gly 1285 1290 1295 aag ctg gag aag gct gtt ctc tct tca gca ggc ttg tct gaa gca tct 3936 Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser 1300 1305 1310 ggc aaa gct gag ttt ctt cct aaa gtt cga gtt cat cgg gaa gac ctg 3984 Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu 1315 1320 1325 ttg cct caa aaa acc agc aat gtt tct tgc gca cac ggg gat ctc ggc 4032 Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly 1330 1335 1340 cag gag atc ttc ctg cag aaa aca cgg gga cct gtt aac ctg aac aaa 4080 Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys 1345 1350 1355 1360 gta aat aga cct gga agg act ccc tcc aag ctt ctg ggt ccc ccg atg 4128 Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met 1365 1370 1375 ccc aaa gag tgg gaa tcc cta gag aag tca cca aaa agc aca gct ctc 4176 Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu 1380 1385 1390 agg acg aaa gac atc atc agt tta ccc ctg gac cgt cac gaa agc aat 4224 Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn 1395 1400 1405 cat tca ata gca gca aaa aat gaa gga caa gcc gag acc caa aga gaa 4272 His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu 1410 1415 1420 gcc gcc tgg acg aag cag gga ggg cct gga agg ctg tgc gct cca aag 4320 Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys 1425 1430 1435 1440 cct ccg gtc ctg cga cgg cat cag agg gac ata agc ctt cct act ttt 4368 Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe 1445 1450 1455 cag ccg gag gaa gac aaa atg gac tat gat gat atc ttc tca act gaa 4416 Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu 1460 1465 1470 acg aag gga gaa gat ttt gac att tac ggt gag gat gaa aat cag gac 4464 Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp 1475 1480 1485 cct cgc agc ttt cag aag aga acc cga cac tat ttc att gct gcg gtg 4512 Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val 1490 1495 1500 gag cag ctc tgg gat tac ggg atg agc gaa tcc ccc cgg gcg cta aga 4560 Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg 1505 1510 1515 1520 aac agg gct cag aac gga gag gtg cct cgg ttc aag aag gtg gtc ttc 4608 Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe 1525 1530 1535 cgg gaa ttt gct gac ggc tcc ttc acg cag ccg tcg tac cgc ggg gaa 4656 Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu 1540 1545 1550 ctc aac aaa cac ttg ggg ctc ttg gga ccc tac atc aga gcg gaa gtt 4704 Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val 1555 1560 1565 gaa gac aac atc atg gta act ttc aaa aac cag gcg tct cgt ccc tat 4752 Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr 1570 1575 1580 tcc ttc tac tcg agc ctt att tct tat ccg gat gat cag gag caa ggg 4800 Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly 1585 1590 1595 1600 gca gaa cct cga cac aac ttc gtc cag cca aat gaa acc aga act tac 4848 Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr 1605 1610 1615 ttt tgg aaa gtg cag cat cac atg gca ccc aca gaa gac gag ttt gac 4896 Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp 1620 1625 1630 tgc aaa gcc tgg gcc tac ttt tct gat gtt gac ctg gaa aaa gat gtg 4944 Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val 1635 1640 1645 cac tca ggc ttg atc ggc ccc ctt ctg atc tgc cgc gcc aac acc ctg 4992 His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu 1650 1655 1660 aac gct gct cac ggt aga caa gtg acc gtg caa gaa ttt gct ctg ttt 5040 Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe 1665 1670 1675 1680 ttc act att ttt gat gag aca aag agc tgg tac ttc act gaa aat gtg 5088 Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val 1685 1690 1695 gaa agg aac tgc cgg gcc ccc tgc cac ctg cag atg gag gac ccc act 5136 Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr 1700 1705 1710 ctg aaa gaa aac tat cgc ttc cat gca atc aat ggc tat gtg atg gat 5184 Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp 1715 1720 1725 aca ctc cct ggc tta gta atg gct cag aat caa agg atc cga tgg tat 5232 Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr 1730 1735 1740 ctg ctc agc atg ggc agc aat gaa aat atc cat tcg att cat ttt agc 5280 Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser 1745 1750 1755 1760 gga cac gtg ttc agt gta cgg aaa aag gag gag tat aaa atg gcc gtg 5328 Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val 1765 1770 1775 tac aat ctc tat ccg ggt gtc ttt gag aca gtg gaa atg cta ccg tcc 5376 Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser 1780 1785 1790 aaa gtt gga att tgg cga ata gaa tgc ctg att ggc gag cac ctg caa 5424 Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln 1795 1800 1805 gct ggg atg agc acg act ttc ctg gtg tac agc aag gag tgt cag gct 5472 Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala 1810 1815 1820 cca ctg gga atg gct tct gga cgc att aga gat ttt cag atc aca gct 5520 Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala 1825 1830 1835 1840 tca gga cag tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat 5568 Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr 1845 1850 1855 tcc gga tca atc aat gcc tgg agc acc aag gat ccc cac tcc tgg atc 5616 Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile 1860 1865 1870 aag gtg gat ctg ttg gca cca atg atc att cac ggc atc atg acc cag 5664 Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln 1875 1880 1885 ggt gcc cgt cag aag ttt tcc agc ctc tac atc tcc cag ttt atc atc 5712 Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 1890 1895 1900 atg tac agt ctt gac ggg agg aac tgg cag agt tac cga ggg aat tcc 5760 Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser 1905 1910 1915 1920 acg ggc acc tta atg gtc ttc ttt ggc aat gtg gac gca tct ggg att 5808 Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile 1925 1930 1935 aaa cac aat att ttt aac cct ccg att gtg gct cgg tac atc cgt ttg 5856 Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu 1940 1945 1950 cac cca aca cat tac agc atc cgc agc act ctt cgc atg gag ttg atg 5904 His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met 1955 1960 1965 ggc tgt gat tta aac agt tgc agc atg ccc ctg gga atg cag aat aaa 5952 Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys 1970 1975 1980 gcg ata tca gac tca cag atc acg gcc tcc tcc cac cta agc aat ata 6000 Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile 1985 1990 1995 2000 ttt gcc acc tgg tct cct tca caa gcc cga ctt cac ctc cag ggg cgg 6048 Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg 2005 2010 2015 acg aat gcc tgg cga ccc cgg gtg agc agc gca gag gag tgg ctg cag 6096 Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln 2020 2025 2030 gtg gac ctg cag aag acg gtg aag gtc aca ggc atc acc acc cag ggc 6144 Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly 2035 2040 2045 gtg aag tcc ctg ctc agc agc atg tat gtg aag gag ttc ctc gtg tcc 6192 Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser 2050 2055 2060 agt agt cag gac ggc cgc cgc tgg acc ctg ttt ctt cag gac ggc cac 6240 Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His 2065 2070 2075 2080 acg aag gtt ttt cag ggc aat cag gac tcc tcc acc ccc gtg gtg aac 6288 Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn 2085 2090 2095 gct ctg gac ccc ccg ctg ttc acg cgc tac ctg agg atc cac ccc acg 6336 Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr 2100 2105 2110 agc tgg gcg cag cac atc gcc ctg agg ctc gag gtt cta gga tgt gag 6384 Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu 2115 2120 2125 gca cag gat ctc tac tga 6402 Ala Gln Asp Leu Tyr 2130 2 2133 PRT Sus scrofa 2 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp 770 775 780 Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu 785 790 795 800 Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro 805 810 815 Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu 820 825 830 Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser 835 840 845 Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val 850 855 860 Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met 865 870 875 880 Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp 885 890 895 Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His 900 905 910 Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys 915 920 925 Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr Glu 930 935 940 Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser 945 950 955 960 Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr 965 970 975 Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn 980 985 990 Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp 995 1000 1005 Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys 1010 1015 1020 Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly 1025 1030 1035 1040 Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu 1045 1050 1055 Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser 1060 1065 1070 Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys 1075 1080 1085 Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn 1090 1095 1100 Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser 1105 1110 1115 1120 Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp 1125 1130 1135 Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn 1140 1145 1150 Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly 1155 1160 1165 Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Ser Ala 1170 1175 1180 Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu 1185 1190 1195 1200 Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser 1205 1210 1215 Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro 1220 1225 1230 Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser 1235 1240 1245 Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn 1250 1255 1260 Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly 1265 1270 1275 1280 Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser Gly 1285 1290 1295 Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser 1300 1305 1310 Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu 1315 1320 1325 Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly 1330 1335 1340 Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys 1345 1350 1355 1360 Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met 1365 1370 1375 Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu 1380 1385 1390 Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn 1395 1400 1405 His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu 1410 1415 1420 Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys 1425 1430 1435 1440 Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe 1445 1450 1455 Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu 1460 1465 1470 Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp 1475 1480 1485 Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val 1490 1495 1500 Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg 1505 1510 1515 1520 Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe 1525 1530 1535 Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu 1540 1545 1550 Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val 1555 1560 1565 Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr 1570 1575 1580 Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly 1585 1590 1595 1600 Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr 1605 1610 1615 Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp 1620 1625 1630 Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val 1635 1640 1645 His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu 1650 1655 1660 Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe 1665 1670 1675 1680 Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val 1685 1690 1695 Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr 1700 1705 1710 Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp 1715 1720 1725 Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr 1730 1735 1740 Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser 1745 1750 1755 1760 Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val 1765 1770 1775 Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser 1780 1785 1790 Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln 1795 1800 1805 Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala 1810 1815 1820 Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala 1825 1830 1835 1840 Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr 1845 1850 1855 Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile 1860 1865 1870 Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln 1875 1880 1885 Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 1890 1895 1900 Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser 1905 1910 1915 1920 Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile 1925 1930 1935 Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu 1940 1945 1950 His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met 1955 1960 1965 Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys 1970 1975 1980 Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile 1985 1990 1995 2000 Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg 2005 2010 2015 Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln 2020 2025 2030 Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly 2035 2040 2045 Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser 2050 2055 2060 Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His 2065 2070 2075 2080 Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn 2085 2090 2095 Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr 2100 2105 2110 Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu 2115 2120 2125 Ala Gln Asp Leu Tyr 2130 3 4404 DNA Sus scrofa gene (1)...(4401) Factor VIII-- B-domain deleted 3 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt ctc tgg gtc ctt ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 aag aat gtc att gaa cct agg agc ttt gcc cag aat tca aga ccc cct 2304 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 agt gcg agc gct cca aag cct ccg gtc ctg cga cgg cat cag agg gac 2352 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 ata agc ctt cct act ttt cag ccg gag gaa gac aaa atg gac tat gat 2400 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 gat atc ttc tca act gaa acg aag gga gaa gat ttt gac att tac ggt 2448 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 gag gat gaa aat cag gac cct cgc agc ttt cag aag aga acc cga cac 2496 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 gat gat cag gag caa ggg gca gaa cct cga cac aac ttc gtc cag cca 2832 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 aat gaa acc aga act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 gtg gaa atg cta ccg tcc aaa gtt gga att tgg cga ata gaa tgc ctg 3408 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 agc aag gag tgt cag gct cca ctg gga atg gct tct gga cgc att aga 3504 Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg 1155 1160 1165 gat ttt cag atc aca gct tca gga cag tat gga cag tgg gcc cca aag 3552 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 gat ccc cac tcc tgg atc aag gtg gat ctg ttg gca cca atg atc att 3648 Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 cac ggc atc atg acc cag ggt gcc cgt cag aag ttt tcc agc ctc tac 3696 His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 atc tcc cag ttt atc atc atg tac agt ctt gac ggg agg aac tgg cag 3744 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln 1235 1240 1245 agt tac cga ggg aat tcc acg ggc acc tta atg gtc ttc ttt ggc aat 3792 Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 gtg gac gca tct ggg att aaa cac aat att ttt aac cct ccg att gtg 3840 Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val 1265 1270 1275 1280 gct cgg tac atc cgt ttg cac cca aca cat tac agc atc cgc agc act 3888 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 ctt cgc atg gag ttg atg ggc tgt gat tta aac agt tgc agc atg ccc 3936 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 ctg gga atg cag aat aaa gcg ata tca gac tca cag atc acg gcc tcc 3984 Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser 1315 1320 1325 tcc cac cta agc aat ata ttt gcc acc tgg tct cct tca caa gcc cga 4032 Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg 1330 1335 1340 ctt cac ctc cag ggg cgg acg aat gcc tgg cga ccc cgg gtg agc agc 4080 Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser 1345 1350 1355 1360 gca gag gag tgg ctg cag gtg gac ctg cag aag acg gtg aag gtc aca 4128 Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr 1365 1370 1375 ggc atc acc acc cag ggc gtg aag tcc ctg ctc agc agc atg tat gtg 4176 Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val 1380 1385 1390 aag gag ttc ctc gtg tcc agt agt cag gac ggc cgc cgc tgg acc ctg 4224 Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu 1395 1400 1405 ttt ctt cag gac ggc cac acg aag gtt ttt cag ggc aat cag gac tcc 4272 Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 tcc acc ccc gtg gtg aac gct ctg gac ccc ccg ctg ttc acg cgc tac 4320 Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr 1425 1430 1435 1440 ctg agg atc cac ccc acg agc tgg gcg cag cac atc gcc ctg agg ctc 4368 Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu 1445 1450 1455 gag gtt cta gga tgt gag gca cag gat ctc tac tga 4404 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 4 1467 PRT Sus scrofa 4 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg 1155 1160 1165 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln 1235 1240 1245 Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val 1265 1270 1275 1280 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser 1315 1320 1325 Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg 1330 1335 1340 Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser 1345 1350 1355 1360 Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr 1365 1370 1375 Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val 1380 1385 1390 Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu 1395 1400 1405 Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr 1425 1430 1435 1440 Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu 1445 1450 1455 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 5 9009 DNA Homo sapiens gene (0)...(0) Factor VIII- Full Length 5 cagtgggtaa gttccttaaa tgctctgcaa agaaattggg acttttcatt aaatcagaaa 60 ttttactttt ttcccctcct gggagctaaa gatattttag agaagaatta accttttgct 120 tctccagttg aacatttgta gcaataagtc atgcaaatag agctctccac ctgcttcttt 180 ctgtgccttt tgcgattctg ctttagt gcc acc aga aga tac tac ctg ggt gca 234 Ala Thr Arg Arg Tyr Tyr Leu Gly Ala 1 5 gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 282 Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro 10 15 20 25 gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 330 Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn 30 35 40 acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gtt cac 378 Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His 45 50 55 ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 426 Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly 60 65 70 cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 474 Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys 75 80 85 aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 522 Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr 90 95 100 105 tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 570 Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg 110 115 120 gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 618 Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val 125 130 135 tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 666 Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys 140 145 150 ctt acc tac tca tat ctt tct cat gtg gac ctg gta aaa gac ttg aat 714 Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn 155 160 165 tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 762 Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala 170 175 180 185 aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 810 Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val 190 195 200 ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 858 Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met 205 210 215 cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 906 Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr 220 225 230 gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 954 Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His 235 240 245 agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1002 Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu 250 255 260 265 gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1050 Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His 270 275 280 cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1098 Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln 285 290 295 aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1146 Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser 300 305 310 tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1194 Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys 315 320 325 cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1242 Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp 330 335 340 345 tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1290 Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp 350 355 360 gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1338 Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys 365 370 375 cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1386 His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp 380 385 390 gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1434 Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser 395 400 405 caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 1482 Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys 410 415 420 425 gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 1530 Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala 430 435 440 att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 1578 Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val 445 450 455 gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 1626 Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr 460 465 470 aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 1674 Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg 475 480 485 aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 1722 Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro 490 495 500 505 gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 1770 Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro 510 515 520 act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 1818 Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val 525 530 535 aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 1866 Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile 540 545 550 tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 1914 Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp 555 560 565 aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 1962 Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp 570 575 580 585 tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2010 Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val 590 595 600 cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2058 Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile 605 610 615 aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2106 Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu 620 625 630 gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2154 Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu 635 640 645 tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2202 Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu 650 655 660 665 gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2250 Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser 670 675 680 atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2298 Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe 685 690 695 cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2346 Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys 700 705 710 aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2394 Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr 715 720 725 ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2442 Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn 730 735 740 745 tca aga cac cct agc act agg caa aag caa ttt aat gcc acc aca att 2490 Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile 750 755 760 cca gaa aat gac ata gag aag act gac cct tgg ttt gca cac aga aca 2538 Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr 765 770 775 cct atg cct aaa ata caa aat gtc tcc tct agt gat ttg ttg atg ctc 2586 Pro Met Pro Lys Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu 780 785 790 ttg cga cag agt cct act cca cat ggg cta tcc tta tct gat ctc caa 2634 Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln 795 800 805 gaa gcc aaa tat gag act ttt tct gat gat cca tca cct gga gca ata 2682 Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile 810 815 820 825 gac agt aat aac agc ctg tct gaa atg aca cac ttc agg cca cag ctc 2730 Asp Ser Asn Asn Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu 830 835 840 cat cac agt ggg gac atg gta ttt acc cct gag tca ggc ctc caa tta 2778 His His Ser Gly Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu 845 850 855 aga tta aat gag aaa ctg ggg aca act gca gca aca gag ttg aag aaa 2826 Arg Leu Asn Glu Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys 860 865 870 ctt gat ttc aaa gtt tct agt aca tca aat aat ctg att tca aca att 2874 Leu Asp Phe Lys Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile 875 880 885 cca tca gac aat ttg gca gca ggt act gat aat aca agt tcc tta gga 2922 Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly 890 895 900 905 ccc cca agt atg cca gtt cat tat gat agt caa tta gat acc act cta 2970 Pro Pro Ser Met Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu 910 915 920 ttt ggc aaa aag tca tct ccc ctt act gag tct ggt gga cct ctg agc 3018 Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser 925 930 935 ttg agt gaa gaa aat aat gat tca aag ttg tta gaa tca ggt tta atg 3066 Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met 940 945 950 aat agc caa gaa agt tca tgg gga aaa aat gta tcg tca aca gag agt 3114 Asn Ser Gln Glu Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser 955 960 965 ggt agg tta ttt aaa ggg aaa aga gct cat gga cct gct ttg ttg act 3162 Gly Arg Leu Phe Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr 970 975 980 985 aaa gat aat gcc tta ttc aaa gtt agc atc tct ttg tta aag aca aac 3210 Lys Asp Asn Ala Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn 990 995 1000 aaa act tcc aat aat tca gca act aat aga aag act cac att gat ggc 3258 Lys Thr Ser Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly 1005 1010 1015 cca tca tta tta att gag aat agt cca tca gtc tgg caa aat ata tta 3306 Pro Ser Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu 1020 1025 1030 gaa agt gac act gag ttt aaa aaa gtg aca cct ttg att cat gac aga 3354 Glu Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg 1035 1040 1045 atg ctt atg gac aaa aat gct aca gct ttg agg cta aat cat atg tca 3402 Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser 1050 1055 1060 1065 aat aaa act act tca tca aaa aac atg gaa atg gtc caa cag aaa aaa 3450 Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys 1070 1075 1080 gag ggc ccc att cca cca gat gca caa aat cca gat atg tcg ttc ttt 3498 Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe 1085 1090 1095 aag atg cta ttc ttg cca gaa tca gca agg tgg ata caa agg act cat 3546 Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110 gga aag aac tct ctg aac tct ggg caa ggc ccc agt cca aag caa tta 3594 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln Leu 1115 1120 1125 gta tcc tta gga cca gaa aaa tct gtg gaa ggt cag aat ttc ttg tct 3642 Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe Leu Ser 1130 1135 1140 1145 gag aaa aac aaa gtg gta gta gga aag ggt gaa ttt aca aag gac gta 3690 Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr Lys Asp Val 1150 1155 1160 gga ctc aaa gag atg gtt ttt cca agc agc aga aac cta ttt ctt act 3738 Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn Leu Phe Leu Thr 1165 1170 1175 aac ttg gat aat tta cat gaa aat aat aca cac aat caa gaa aaa aaa 3786 Asn Leu Asp Asn Leu His Glu Asn Asn Thr His Asn Gln Glu Lys Lys 1180 1185 1190 att cag gaa gaa ata gaa aag aag gaa aca tta atc caa gag aat gta 3834 Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr Leu Ile Gln Glu Asn Val 1195 1200 1205 gtt ttg cct cag ata cat aca gtg act ggc act aag aat ttc atg aag 3882 Val Leu Pro Gln Ile His Thr Val Thr Gly Thr Lys Asn Phe Met Lys 1210 1215 1220 1225 aac ctt ttc tta ctg agc act agg caa aat gta gaa ggt tca tat gag 3930 Asn Leu Phe Leu Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu 1230 1235 1240 ggg gca tat gct cca gta ctt caa gat ttt agg tca tta aat gat tca 3978 Gly Ala Tyr Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser 1245 1250 1255 aca aat aga aca aag aaa cac aca gct cat ttc tca aaa aaa ggg gag 4026 Thr Asn Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu 1260 1265 1270 gaa gaa aac ttg gaa ggc ttg gga aat caa acc aag caa att gta gag 4074 Glu Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu 1275 1280 1285 aaa tat gca tgc acc aca agg ata tct cct aat aca agc cag cag aat 4122 Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn 1290 1295 1300 1305 ttt gtc acg caa cgt agt aag aga gct ttg aaa caa ttc aga ctc cca 4170 Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro 1310 1315 1320 cta gaa gaa aca gaa ctt gaa aaa agg ata att gtg gat gac acc tca 4218 Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser 1325 1330 1335 acc cag tgg tcc aaa aac atg aaa cat ttg acc ccg agc acc ctc aca 4266 Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 cag ata gac tac aat gag aag gag aaa ggg gcc att act cag tct ccc 4314 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser Pro 1355 1360 1365 tta tca gat tgc ctt acg agg agt cat agc atc cct caa gca aat aga 4362 Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala Asn Arg 1370 1375 1380 1385 tct cca tta ccc att gca aag gta tca tca ttt cca tct att aga cct 4410 Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser Ile Arg Pro 1390 1395 1400 ata tat ctg acc agg gtc cta ttc caa gac aac tct tct cat ctt cca 4458 Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser Ser His Leu Pro 1405 1410 1415 gca gca tct tat aga aag aaa gat tct ggg gtc caa gaa agc agt cat 4506 Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val Gln Glu Ser Ser His 1420 1425 1430 ttc tta caa gga gcc aaa aaa aat aac ctt tct tta gcc att cta acc 4554 Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr 1435 1440 1445 ttg gag atg act ggt gat caa aga gag gtt ggc tcc ctg ggg aca agt 4602 Leu Glu Met Thr Gly Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser 1450 1455 1460 1465 gcc aca aat tca gtc aca tac aag aaa gtt gag aac act gtt ctc ccg 4650 Ala Thr Asn Ser Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro 1470 1475 1480 aaa cca gac ttg ccc aaa aca tct ggc aaa gtt gaa ttg ctt cca aaa 4698 Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys 1485 1490 1495 gtt cac att tat cag aag gac cta ttc cct acg gaa act agc aat ggg 4746 Val His Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly 1500 1505 1510 tct cct ggc cat ctg gat ctc gtg gaa ggg agc ctt ctt cag gga aca 4794 Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1515 1520 1525 gag gga gcg att aag tgg aat gaa gca aac aga cct gga aaa gtt ccc 4842 Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro 1530 1535 1540 1545 ttt ctg aga gta gca aca gaa agc tct gca aag act ccc tcc aag cta 4890 Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu 1550 1555 1560 ttg gat cct ctt gct tgg gat aac cac tat ggt act cag ata cca aaa 4938 Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys 1565 1570 1575 gaa gag tgg aaa tcc caa gag aag tca cca gaa aaa aca gct ttt aag 4986 Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 aaa aag gat acc att ttg tcc ctg aac gct tgt gaa agc aat cat gca 5034 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His Ala 1595 1600 1605 ata gca gca ata aat gag gga caa aat aag ccc gaa ata gaa gtc acc 5082 Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu Val Thr 1610 1615 1620 1625 tgg gca aag caa ggt agg act gaa agg ctg tgc tct caa aac cca cca 5130 Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln Asn Pro Pro 1630 1635 1640 gtc ttg aaa cgc cat caa cgg gaa ata act cgt act act ctt cag tca 5178 Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser 1645 1650 1655 gat caa gag gaa att gac tat gat gat acc ata tca gtt gaa atg aag 5226 Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys 1660 1665 1670 aag gaa gat ttt gac att tat gat gag gat gaa aat cag agc ccc cgc 5274 Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg 1675 1680 1685 agc ttt caa aag aaa aca cga cac tat ttt att gct gca gtg gag agg 5322 Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg 1690 1695 1700 1705 ctc tgg gat tat ggg atg agt agc tcc cca cat gtt cta aga aac agg 5370 Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg 1710 1715 1720 gct cag agt ggc agt gtc cct cag ttc aag aaa gtt gtt ttc cag gaa 5418 Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu 1725 1730 1735 ttt act gat ggc tcc ttt act cag ccc tta tac cgt gga gaa cta aat 5466 Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn 1740 1745 1750 gaa cat ttg gga ctc ctg ggg cca tat ata aga gca gaa gtt gaa gat 5514 Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1755 1760 1765 aat atc atg gta act ttc aga aat cag gcc tct cgt ccc tat tcc ttc 5562 Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe 1770 1775 1780 1785 tat tct agc ctt att tct tat gag gaa gat cag agg caa gga gca gaa 5610 Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu 1790 1795 1800 cct aga aaa aac ttt gtc aag cct aat gaa acc aaa act tac ttt tgg 5658 Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp 1805 1810 1815 aaa gtg caa cat cat atg gca ccc act aaa gat gag ttt gac tgc aaa 5706 Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830 gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa gat gtg cac tca 5754 Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser 1835 1840 1845 ggc ctg att gga ccc ctt ctg gtc tgc cac act aac aca ctg aac cct 5802 Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro 1850 1855 1860 1865 gct cat ggg aga caa gtg aca gta cag gaa ttt gct ctg ttt ttc acc 5850 Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr 1870 1875 1880 atc ttt gat gag acc aaa agc tgg tac ttc act gaa aat atg gaa aga 5898 Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg 1885 1890 1895 aac tgc agg gct ccc tgc aat atc cag atg gaa gat ccc act ttt aaa 5946 Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1900 1905 1910 gag aat tat cgc ttc cat gca atc aat ggc tac ata atg gat aca cta 5994 Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu 1915 1920 1925 cct ggc tta gta atg gct cag gat caa agg att cga tgg tat ctg ctc 6042 Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu 1930 1935 1940 1945 agc atg ggc agc aat gaa aac atc cat tct att cat ttc agt gga cat 6090 Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His 1950 1955 1960 gtg ttc act gta cga aaa aaa gag gag tat aaa atg gca ctg tac aat 6138 Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn 1965 1970 1975 ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta cca tcc aaa gct 6186 Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala 1980 1985 1990 gga att tgg cgg gtg gaa tgc ctt att ggc gag cat cta cat gct ggg 6234 Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 1995 2000 2005 atg agc aca ctt ttt ctg gtg tac agc aat aag tgt cag act ccc ctg 6282 Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu 2010 2015 2020 2025 gga atg gct tct gga cac att aga gat ttt cag att aca gct tca gga 6330 Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly 2030 2035 2040 caa tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat tcc gga 6378 Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly 2045 2050 2055 tca atc aat gcc tgg agc acc aag gag ccc ttt tct tgg atc aag gtg 6426 Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070 gat ctg ttg gca cca atg att att cac ggc atc aag acc cag ggt gcc 6474 Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala 2075 2080 2085 cgt cag aag ttc tcc agc ctc tac atc tct cag ttt atc atc atg tat 6522 Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr 2090 2095 2100 2105 agt ctt gat ggg aag aag tgg cag act tat cga gga aat tcc act gga 6570 Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly 2110 2115 2120 acc tta atg gtc ttc ttt ggc aat gtg gat tca tct ggg ata aaa cac 6618 Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His 2125 2130 2135 aat att ttt aac cct cca att att gct cga tac atc cgt ttg cac cca 6666 Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 2140 2145 2150 act cat tat agc att cgc agc act ctt cgc atg gag ttg atg ggc tgt 6714 Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys 2155 2160 2165 gat tta aat agt tgc agc atg cca ttg gga atg gag agt aaa gca ata 6762 Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile 2170 2175 2180 2185 tca gat gca cag att act gct tca tcc tac ttt acc aat atg ttt gcc 6810 Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala 2190 2195 2200 acc tgg tct cct tca aaa gct cga ctt cac ctc caa ggg agg agt aat 6858 Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn 2205 2210 2215 gcc tgg aga cct cag gtg aat aat cca aaa gag tgg ctg caa gtg gac 6906 Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp 2220 2225 2230 ttc cag aag aca atg aaa gtc aca gga gta act act cag gga gta aaa 6954 Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2235 2240 2245 tct ctg ctt acc agc atg tat gtg aag gag ttc ctc atc tcc agc agt 7002 Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser 2250 2255 2260 2265 caa gat ggc cat cag tgg act ctc ttt ttt cag aat ggc aaa gta aag 7050 Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys 2270 2275 2280 gtt ttt cag gga aat caa gac tcc ttc aca cct gtg gtg aac tct cta 7098 Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu 2285 2290 2295 gac cca ccg tta ctg act cgc tac ctt cga att cac ccc cag agt tgg 7146 Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310 gtg cac cag att gcc ctg agg atg gag gtt ctg ggc tgc gag gca cag 7194 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln 2315 2320 2325 gac ctc tac tgagggtggc cactgcagca cctgccactg ccgtcacctc 7243 Asp Leu Tyr 2330 tccctcctca gctccagggc agtgtccctc cctggcttgc cttctacctt tgtgctaaat 7303 cctagcagac actgccttga agcctcctga attaactatc atcagtcctg catttctttg 7363 gtggggggcc aggagggtgc atccaattta acttaactct tacctatttt ctgcagctgc 7423 tcccagatta ctccttcctt ccaatataac taggcaaaaa gaagtgagga gaaacctgca 7483 tgaaagcatt cttccctgaa aagttaggcc tctcagagtc accacttcct ctgttgtaga 7543 aaaactatgt gatgaaactt tgaaaaagat atttatgatg ttaacatttc aggttaagcc 7603 tcatacgttt aaaataaaac tctcagttgt ttattatcct gatcaagcat ggaacaaagc 7663 atgtttcagg atcagatcaa tacaatcttg gagtcaaaag gcaaatcatt tggacaatct 7723 gcaaaatgga gagaatacaa taactactac agtaaagtct gtttctgctt ccttacacat 7783 agatataatt atgttattta gtcattatga ggggcacatt cttatctcca aaactagcat 7843 tcttaaactg agaattatag atggggttca agaatcccta agtcccctga aattatataa 7903 ggcattctgt ataaatgcaa atgtgcattt ttctgacgag tgtccataga tataaagcca 7963 ttggtcttaa ttctgaccaa taaaaaaata agtcaggagg atgcaattgt tgaaagcttt 8023 gaaataaaat aacatgtctt cttgaaattt gtgatggcca agaaagaaaa tgatgatgac 8083 attaggcttc taaaggacat acatttaata tttctgtgga aatatgagga aaatccatgg 8143 ttatctgaga taggagatac aaactttgta attctaataa tgcactcagt ttactctctc 8203 cctctactaa tttcctgctg aaaataacac aacaaaaatg taacagggga aattatatac 8263 cgtgactgaa aactagagtc ctacttacat agttgaaata tcaaggaggt cagaagaaaa 8323 ttggactggt gaaaacagaa aaaacactcc agtctgccat atcaccacac aataggatcc 8383 cccttcttgc cctccacccc cataagattg tgaagggttt actgctcctt ccatctgcct 8443 gcaccccttc actatgacta cacagaactc tcctgatagt aaagggggct ggaggcaagg 8503 ataagttata gagcagttgg aggaagcatc caaagactgc aacccagggc aaatggaaaa 8563 caggagatcc taatatgaaa gaaaaatgga tcccaatctg agaaaaggca aaagaatggc 8623 tacttttttc tatgctggag tattttctaa taatcctgct tgacccttat ctgacctctt 8683 tggaaactat aacatagctg tcacagtata gtcacaatcc acaaatgatg caggtgcaaa 8743 tggtttatag ccctgtgaag ttcttaaagt ttagaggcta acttacagaa atgaataagt 8803 tgttttgttt tatagcccgg tagaggagtt aaccccaaag gtgatatggt tttatttcct 8863 gttatgttta acttgataat cttattttgg cattcttttc ccattgacta tatacatctc 8923 tatttctcaa atgttcatgg aactagctct tttattttcc tgctggtttc ttcagtaatg 8983 agttaaataa aacattgaca cataca 9009 6 2351 PRT Homo sapiens 6 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365 Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765 Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp 770 775 780 Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys 785 790 795 800 Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser 805 810 815 Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830 Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840 845 Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly 850 855 860 Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu 865 870 875 880 Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys 885 890 895 Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn 900 905 910 Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925 Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940 Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu 945 950 955 960 Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu 965 970 975 Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe 980 985 990 Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala 995 1000 1005 Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser 1010 1015 1020 Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser 1025 1030 1035 Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu 1040 1045 1050 Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg 1055 1060 1065 Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met 1070 1075 1080 Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln 1085 1090 1095 Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met 1100 1105 1110 Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile 1115 1120 1125 Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro 1130 1135 1140 Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu 1145 1150 1155 Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys 1160 1165 1170 Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro 1175 1180 1185 Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1190 1195 1200 Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu 1205 1210 1215 Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile 1220 1225 1230 His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu 1235 1240 1245 Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr 1250 1255 1260 Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn 1265 1270 1275 Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu 1280 1285 1290 Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu 1295 1300 1305 Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln 1310 1315 1320 Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg 1325 1330 1335 Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp 1340 1345 1350 Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro 1355 1360 1365 Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala 1370 1375 1380 Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser 1385 1390 1395 Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser 1400 1405 1410 Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe 1415 1420 1425 Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1430 1435 1440 Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys 1445 1450 1455 Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly 1460 1465 1470 Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser 1475 1480 1485 Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp 1490 1495 1500 Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His 1505 1510 1515 Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser 1520 1525 1530 Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1535 1540 1545 Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val 1550 1555 1560 Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser 1565 1570 1575 Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln 1580 1585 1590 Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys 1595 1600 1605 Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys 1610 1615 1620 Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys 1625 1630 1635 Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg 1640 1645 1650 Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 1655 1660 1665 Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1670 1675 1680 Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1685 1690 1695 Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1700 1705 1710 Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1715 1720 1725 Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1730 1735 1740 Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1745 1750 1755 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1760 1765 1770 His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1775 1780 1785 Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1790 1795 1800 Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1805 1810 1815 Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1820 1825 1830 Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1835 1840 1845 Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1850 1855 1860 Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1865 1870 1875 Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1880 1885 1890 Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1895 1900 1905 Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1910 1915 1920 Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1925 1930 1935 Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1940 1945 1950 Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1955 1960 1965 Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1970 1975 1980 Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1985 1990 1995 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 2000 2005 2010 Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 2015 2020 2025 Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 2030 2035 2040 Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 2045 2050 2055 Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 2060 2065 2070 Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 2075 2080 2085 Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 2090 2095 2100 Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 2105 2110 2115 Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 2120 2125 2130 Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 2135 2140 2145 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2150 2155 2160 Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 2165 2170 2175 Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 2180 2185 2190 Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 2195 2200 2205 Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 2210 2215 2220 Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 2225 2230 2235 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 2240 2245 2250 Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2255 2260 2265 Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 2270 2275 2280 Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 2285 2290 2295 Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 2300 2305 2310 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 2315 2320 2325 Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 2330 2335 2340 Gly Cys Glu Ala Gln Asp Leu Tyr 2345 2350 7 6996 DNA Homo sapiens gene (0)...(0) Factor VIII- Full Length coding sequence 7 gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat 60 ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc 120 aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggttca ccttttcaac 180 atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt 240 tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct 300 gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa 360 agggagaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc 420 ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct 480 catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga 540 gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct 600 gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg 660 gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg 720 tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg 780 ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac 840 catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg 900 atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg 960 gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat 1020 gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt 1080 gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa 1140 acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc 1200 gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt 1260 aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa 1320 gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca 1380 ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc 1440 actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat 1500 tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg 1560 ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag 1620 agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat 1680 caaagaggaa accagataat gtcagacaag aggaatgtca tcctgttttc tgtatttgat 1740 gagaaccgaa gctggtacct cacagagaat atacaacgct ttctccccaa tccagctgga 1800 gtgcagcttg aggatccaga gttccaagcc tccaacatca tgcacagcat caatggctat 1860 gtttttgata gtttgcagtt gtcagtttgt ttgcatgagg tggcatactg gtacattcta 1920 agcattggag cacagactga cttcctttct gtcttcttct ctggatatac cttcaaacac 1980 aaaatggtct atgaagacac actcacccta ttcccattct caggagaaac tgtcttcatg 2040 tcgatggaaa acccaggtct atggattctg gggtgccaca actcagactt tcggaacaga 2100 ggcatgaccg ccttactgaa ggtttctagt tgtgacaaga acactggtga ttattacgag 2160 gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga 2220 agcttctccc agaattcaag acaccctagc actaggcaaa agcaatttaa tgccaccaca 2280 attccagaaa atgacataga gaagactgac ccttggtttg cacacagaac acctatgcct 2340 aaaatacaaa atgtctcctc tagtgatttg ttgatgctct tgcgacagag tcctactcca 2400 catgggctat ccttatctga tctccaagaa gccaaatatg agactttttc tgatgatcca 2460 tcacctggag caatagacag taataacagc ctgtctgaaa tgacacactt caggccacag 2520 ctccatcaca gtggggacat ggtatttacc cctgagtcag gcctccaatt aagattaaat 2580 gagaaactgg ggacaactgc agcaacagag ttgaagaaac ttgatttcaa agtttctagt 2640 acatcaaata atctgatttc aacaattcca tcagacaatt tggcagcagg tactgataat 2700 acaagttcct taggaccccc aagtatgcca gttcattatg atagtcaatt agataccact 2760 ctatttggca aaaagtcatc tccccttact gagtctggtg gacctctgag cttgagtgaa 2820 gaaaataatg attcaaagtt gttagaatca ggtttaatga atagccaaga aagttcatgg 2880 ggaaaaaatg tatcgtcaac agagagtggt aggttattta aagggaaaag agctcatgga 2940 cctgctttgt tgactaaaga taatgcctta ttcaaagtta gcatctcttt gttaaagaca 3000 aacaaaactt ccaataattc agcaactaat agaaagactc acattgatgg cccatcatta 3060 ttaattgaga atagtccatc agtctggcaa aatatattag aaagtgacac tgagtttaaa 3120 aaagtgacac ctttgattca tgacagaatg cttatggaca aaaatgctac agctttgagg 3180 ctaaatcata tgtcaaataa aactacttca tcaaaaaaca tggaaatggt ccaacagaaa 3240 aaagagggcc ccattccacc agatgcacaa aatccagata tgtcgttctt taagatgcta 3300 ttcttgccag aatcagcaag gtggatacaa aggactcatg gaaagaactc tctgaactct 3360 gggcaaggcc ccagtccaaa gcaattagta tccttaggac cagaaaaatc tgtggaaggt 3420 cagaatttct tgtctgagaa aaacaaagtg gtagtaggaa agggtgaatt tacaaaggac 3480 gtaggactca aagagatggt ttttccaagc agcagaaacc tatttcttac taacttggat 3540 aatttacatg aaaataatac acacaatcaa gaaaaaaaaa ttcaggaaga aatagaaaag 3600 aaggaaacat taatccaaga gaatgtagtt ttgcctcaga tacatacagt gactggcact 3660 aagaatttca tgaagaacct tttcttactg agcactaggc aaaatgtaga aggttcatat 3720 gagggggcat atgctccagt acttcaagat tttaggtcat taaatgattc aacaaataga 3780 acaaagaaac acacagctca tttctcaaaa aaaggggagg aagaaaactt ggaaggcttg 3840 ggaaatcaaa ccaagcaaat tgtagagaaa tatgcatgca ccacaaggat atctcctaat 3900 acaagccagc agaattttgt cacgcaacgt agtaagagag ctttgaaaca attcagactc 3960 ccactagaag aaacagaact tgaaaaaagg ataattgtgg atgacacctc aacccagtgg 4020 tccaaaaaca tgaaacattt gaccccgagc accctcacac agatagacta caatgagaag 4080 gagaaagggg ccattactca gtctccctta tcagattgcc ttacgaggag tcatagcatc 4140 cctcaagcaa atagatctcc attacccatt gcaaaggtat catcatttcc atctattaga 4200 cctatatatc tgaccagggt cctattccaa gacaactctt ctcatcttcc agcagcatct 4260 tatagaaaga aagattctgg ggtccaagaa agcagtcatt tcttacaagg agccaaaaaa 4320 aataaccttt ctttagccat tctaaccttg gagatgactg gtgatcaaag agaggttggc 4380 tccctgggga caagtgccac aaattcagtc acatacaaga aagttgagaa cactgttctc 4440 ccgaaaccag acttgcccaa aacatctggc aaagttgaat tgcttccaaa agttcacatt 4500 tatcagaagg acctattccc tacggaaact agcaatgggt ctcctggcca tctggatctc 4560 gtggaaggga gccttcttca gggaacagag ggagcgatta agtggaatga agcaaacaga 4620 cctggaaaag ttccctttct gagagtagca acagaaagct ctgcaaagac tccctccaag 4680 ctattggatc ctcttgcttg ggataaccac tatggtactc agataccaaa agaagagtgg 4740 aaatcccaag agaagtcacc agaaaaaaca gcttttaaga aaaaggatac cattttgtcc 4800 ctgaacgctt gtgaaagcaa tcatgcaata gcagcaataa atgagggaca aaataagccc 4860 gaaatagaag tcacctgggc aaagcaaggt aggactgaaa ggctgtgctc tcaaaaccca 4920 ccagtcttga aacgccatca acgggaaata actcgtacta ctcttcagtc agatcaagag 4980 gaaattgact atgatgatac catatcagtt gaaatgaaga aggaagattt tgacatttat 5040 gatgaggatg aaaatcagag cccccgcagc tttcaaaaga aaacacgaca ctattttatt 5100 gctgcagtgg agaggctctg ggattatggg atgagtagct ccccacatgt tctaagaaac 5160 agggctcaga gtggcagtgt ccctcagttc aagaaagttg ttttccagga atttactgat 5220 ggctccttta ctcagccctt ataccgtgga gaactaaatg aacatttggg actcctgggg 5280 ccatatataa gagcagaagt tgaagataat atcatggtaa ctttcagaaa tcaggcctct 5340 cgtccctatt ccttctattc tagccttatt tcttatgagg aagatcagag gcaaggagca 5400 gaacctagaa aaaactttgt caagcctaat gaaaccaaaa cttacttttg gaaagtgcaa 5460 catcatatgg cacccactaa agatgagttt gactgcaaag cctgggctta tttctctgat 5520 gttgacctgg aaaaagatgt gcactcaggc ctgattggac cccttctggt ctgccacact 5580 aacacactga accctgctca tgggagacaa gtgacagtac aggaatttgc tctgtttttc 5640 accatctttg atgagaccaa aagctggtac ttcactgaaa atatggaaag aaactgcagg 5700 gctccctgca atatccagat ggaagatccc acttttaaag agaattatcg cttccatgca 5760 atcaatggct acataatgga tacactacct ggcttagtaa tggctcagga tcaaaggatt 5820 cgatggtatc tgctcagcat gggcagcaat gaaaacatcc attctattca tttcagtgga 5880 catgtgttca ctgtacgaaa aaaagaggag tataaaatgg cactgtacaa tctctatcca 5940 ggtgtttttg agacagtgga aatgttacca tccaaagctg gaatttggcg ggtggaatgc 6000 cttattggcg agcatctaca tgctgggatg agcacacttt ttctggtgta cagcaataag 6060 tgtcagactc ccctgggaat ggcttctgga cacattagag attttcagat tacagcttca 6120 ggacaatatg gacagtgggc cccaaagctg gccagacttc attattccgg atcaatcaat 6180 gcctggagca ccaaggagcc cttttcttgg atcaaggtgg atctgttggc accaatgatt 6240 attcacggca tcaagaccca gggtgcccgt cagaagttct ccagcctcta catctctcag 6300 tttatcatca tgtatagtct tgatgggaag aagtggcaga cttatcgagg aaattccact 6360 ggaaccttaa tggtcttctt tggcaatgtg gattcatctg ggataaaaca caatattttt 6420 aaccctccaa ttattgctcg atacatccgt ttgcacccaa ctcattatag cattcgcagc 6480 actcttcgca tggagttgat gggctgtgat ttaaatagtt gcagcatgcc attgggaatg 6540 gagagtaaag caatatcaga tgcacagatt actgcttcat cctactttac caatatgttt 6600 gccacctggt ctccttcaaa agctcgactt cacctccaag ggaggagtaa tgcctggaga 6660 cctcaggtga ataatccaaa agagtggctg caagtggact tccagaagac aatgaaagtc 6720 acaggagtaa ctactcaggg agtaaaatct ctgcttacca gcatgtatgt gaaggagttc 6780 ctcatctcca gcagtcaaga tggccatcag tggactctct tttttcagaa tggcaaagta 6840 aaggtttttc agggaaatca agactccttc acacctgtgg tgaactctct agacccaccg 6900 ttactgactc gctaccttcg aattcacccc cagagttggg tgcaccagat tgccctgagg 6960 atggaggttc tgggctgcga ggcacaggac ctctac 6996 8 2319 PRT Mus musculus 8 Met Gln Ile Ala Leu Phe Ala Cys Phe Phe Leu Ser Leu Phe Asn Phe 1 5 10 15 Cys Ser Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asn Tyr Ile Gln Ser Asp Leu Leu Ser Val Leu His Thr Asp Ser 35 40 45 Arg Phe Leu Pro Arg Met Ser Thr Ser Phe Pro Phe Asn Thr Ser Ile 50 55 60 Met Tyr Lys Lys Thr Val Phe Val Glu Tyr Lys Asp Gln Leu Phe Asn 65 70 75 80 Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Trp Thr Glu Val His Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala 115 120 125 Ser Glu Gly Asp Glu Tyr Glu Asp Gln Thr Ser Gln Met Glu Lys Glu 130 135 140 Asp Asp Lys Val Phe Pro Gly Glu Ser His Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Met Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Lys Glu Gly Ser Leu Ser Lys Glu Arg 195 200 205 Thr Gln Met Leu Tyr Gln Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Glu Thr Asn Asp Ser Tyr Thr Gln Ser Met 225 230 235 240 Asp Ser Ala Ser Ala Arg Asp Trp Pro Lys Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Ile His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Phe Val Arg Asn His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu 305 310 315 320 Ile Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Lys 325 330 335 His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu 340 345 350 Ser Gln Trp Gln Lys Lys Asn Asn Asn Glu Glu Met Glu Asp Tyr Asp 355 360 365 Asp Asp Leu Tyr Ser Glu Met Asp Met Phe Thr Leu Asp Tyr Asp Ser 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys Tyr Pro Lys Thr 385 390 395 400 Trp Ile His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Ser Val Pro Thr Ser Asp Asn Gly Ser Tyr Lys Ser Gln Tyr Leu Ser 420 425 430 Asn Gly Pro His Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Ile 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Thr Ile Gln His Glu 450 455 460 Ser Gly Leu Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Ser Pro Leu His Ala Arg Arg Leu Pro Arg 500 505 510 Gly Ile Lys His Val Lys Asp Leu Pro Ile His Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Ile Asn Pro Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Ile Phe Asp Glu Asn Gln Ser Trp Tyr Ile Thr Glu 595 600 605 Asn Met Gln Arg Phe Leu Pro Asn Ala Ala Lys Thr Gln Pro Gln Asp 610 615 620 Pro Gly Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Glu Leu Thr Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 His Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Ile Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Phe Arg Lys Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Ser Thr Ser Asp 725 730 735 Tyr Tyr Glu Glu Ile Tyr Glu Asp Ile Pro Thr Gln Leu Val Asn Glu 740 745 750 Asn Asn Val Ile Asp Pro Arg Ser Phe Phe Gln Asn Thr Asn His Pro 755 760 765 Asn Thr Arg Lys Lys Lys Phe Lys Asp Ser Thr Ile Pro Lys Asn Asp 770 775 780 Met Glu Lys Ile Glu Pro Gln Phe Glu Glu Ile Ala Glu Met Leu Lys 785 790 795 800 Val Gln Ser Val Ser Val Ser Asp Met Leu Met Leu Leu Gly Gln Ser 805 810 815 His Pro Thr Pro His Gly Leu Phe Leu Ser Asp Gly Gln Glu Ala Ile 820 825 830 Tyr Glu Ala Ile His Asp Asp His Ser Pro Asn Ala Ile Asp Ser Asn 835 840 845 Glu Gly Pro Ser Lys Val Thr Gln Leu Arg Pro Glu Ser His His Ser 850 855 860 Glu Lys Ile Val Phe Thr Pro Gln Pro Gly Leu Gln Leu Arg Ser Asn 865 870 875 880 Lys Ser Leu Glu Thr Thr Ile Glu Val Lys Trp Lys Lys Leu Gly Leu 885 890 895 Gln Val Ser Ser Leu Pro Ser Asn Leu Met Thr Thr Thr Ile Leu Ser 900 905 910 Asp Asn Leu Lys Ala Thr Phe Glu Lys Thr Asp Ser Ser Gly Phe Pro 915 920 925 Asp Met Pro Val His Ser Ser Ser Lys Leu Ser Thr Thr Ala Phe Gly 930 935 940 Lys Lys Ala Tyr Ser Leu Val Gly Ser His Val Pro Leu Asn Ala Ser 945 950 955 960 Glu Glu Asn Ser Asp Ser Asn Ile Leu Asp Ser Thr Leu Met Tyr Ser 965 970 975 Gln Glu Ser Leu Pro Arg Asp Asn Ile Leu Ser Ile Glu Asn Asp Arg 980 985 990 Leu Leu Arg Glu Lys Arg Phe His Gly Ile Ala Leu Leu Thr Lys Asp 995 1000 1005 Asn Thr Leu Phe Lys Asp Asn Val Ser Leu Met Lys Thr Asn Lys Thr 1010 1015 1020 Tyr Asn His Ser Thr Thr Asn Glu Lys Leu His Thr Glu Ser Pro Thr 1025 1030 1035 1040 Ser Ile Glu Asn Ser Thr Thr Asp Leu Gln Asp Ala Ile Leu Lys Val 1045 1050 1055 Asn Ser Glu Ile Gln Glu Val Thr Ala Leu Ile His Asp Gly Thr Leu 1060 1065 1070 Leu Gly Lys Asn Ser Thr Tyr Leu Arg Leu Asn His Met Leu Asn Arg 1075 1080 1085 Thr Thr Ser Thr Lys Asn Lys Asp Ile Phe His Arg Lys Asp Glu Asp 1090 1095 1100 Pro Ile Pro Gln Asp Glu Glu Asn Thr Ile Met Pro Phe Ser Lys Met 1105 1110 1115 1120 Leu Phe Leu Ser Glu Ser Ser Asn Trp Phe Lys Lys Thr Asn Gly Asn 1125 1130 1135 Asn Ser Leu Asn Ser Glu Gln Glu His Ser Pro Lys Gln Leu Val Tyr 1140 1145 1150 Leu Met Phe Lys Lys Tyr Val Lys Asn Gln Ser Phe Leu Ser Glu Lys 1155 1160 1165 Asn Lys Val Thr Val Glu Gln Asp Gly Phe Thr Lys Asn Ile Gly Leu 1170 1175 1180 Lys Asp Met Ala Phe Pro His Asn Met Ser Ile Phe Leu Thr Thr Leu 1185 1190 1195 1200 Ser Asn Val His Glu Asn Gly Arg His Asn Gln Glu Lys Asn Ile Gln 1205 1210 1215 Glu Glu Ile Glu Lys Glu Ala Leu Ile Glu Glu Lys Val Val Leu Pro 1220 1225 1230 Gln Val His Glu Ala Thr Gly Ser Lys Asn Phe Leu Lys Asp Ile Leu 1235 1240 1245 Ile Leu Gly Thr Arg Gln Asn Ile Ser Leu Tyr Glu Val His Val Pro 1250 1255 1260 Val Leu Gln Asn Ile Thr Ser Ile Asn Asn Ser Thr Asn Thr Val Gln 1265 1270 1275 1280 Ile His Met Glu His Phe Phe Lys Arg Arg Lys Asp Lys Glu Thr Asn 1285 1290 1295 Ser Glu Gly Leu Val Asn Lys Thr Arg Glu Met Val Lys Asn Tyr Pro 1300 1305 1310 Ser Gln Lys Asn Ile Thr Thr Gln Arg Ser Lys Arg Ala Leu Gly Gln 1315 1320 1325 Phe Arg Leu Ser Thr Gln Trp Leu Lys Thr Ile Asn Cys Ser Thr Gln 1330 1335 1340 Cys Ile Ile Lys Gln Ile Asp His Ser Lys Glu Met Lys Lys Phe Ile 1345 1350 1355 1360 Thr Lys Ser Ser Leu Ser Asp Ser Ser Val Ile Lys Ser Thr Thr Gln 1365 1370 1375 Thr Asn Ser Ser Asp Ser His Ile Val Lys Thr Ser Ala Phe Pro Pro 1380 1385 1390 Ile Asp Leu Lys Arg Ser Pro Phe Gln Asn Lys Phe Ser His Val Gln 1395 1400 1405 Ala Ser Ser Tyr Ile Tyr Asp Phe Lys Thr Lys Ser Ser Arg Ile Gln 1410 1415 1420 Glu Ser Asn Asn Phe Leu Lys Glu Thr Lys Ile Asn Asn Pro Ser Leu 1425 1430 1435 1440 Ala Ile Leu Pro Trp Asn Met Phe Ile Asp Gln Gly Lys Phe Thr Ser 1445 1450 1455 Pro Gly Lys Ser Asn Thr Asn Ser Val Thr Tyr Lys Lys Arg Glu Asn 1460 1465 1470 Ile Ile Phe Leu Lys Pro Thr Leu Pro Glu Glu Ser Gly Lys Ile Glu 1475 1480 1485 Leu Leu Pro Gln Val Ser Ile Gln Glu Glu Glu Ile Leu Pro Thr Glu 1490 1495 1500 Thr Ser His Gly Ser Pro Gly His Leu Asn Leu Met Lys Glu Val Phe 1505 1510 1515 1520 Leu Gln Lys Ile Gln Gly Pro Thr Lys Trp Asn Lys Ala Lys Arg His 1525 1530 1535 Gly Glu Ser Ile Lys Gly Lys Thr Glu Ser Ser Lys Asn Thr Arg Ser 1540 1545 1550 Lys Leu Leu Asn His His Ala Trp Asp Tyr His Tyr Ala Ala Gln Ile 1555 1560 1565 Pro Lys Asp Met Trp Lys Ser Lys Glu Lys Ser Pro Glu Ile Ile Ser 1570 1575 1580 Ile Lys Gln Glu Asp Thr Ile Leu Ser Leu Arg Pro His Gly Asn Ser 1585 1590 1595 1600 His Ser Ile Gly Ala Asn Glu Lys Gln Asn Trp Pro Gln Arg Glu Thr 1605 1610 1615 Thr Trp Val Lys Gln Gly Gln Thr Gln Arg Thr Cys Ser Gln Ile Pro 1620 1625 1630 Pro Val Leu Lys Arg His Gln Arg Glu Leu Ser Ala Phe Gln Ser Glu 1635 1640 1645 Gln Glu Ala Thr Asp Tyr Asp Asp Ala Ile Thr Ile Glu Thr Ile Glu 1650 1655 1660 Asp Phe Asp Ile Tyr Ser Glu Asp Ile Lys Gln Gly Pro Arg Ser Phe 1665 1670 1675 1680 Gln Gln Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 1685 1690 1695 Asp Tyr Gly Met Ser Thr Ser His Val Leu Arg Asn Arg Tyr Gln Ser 1700 1705 1710 Asp Asn Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp 1715 1720 1725 Gly Ser Phe Ser Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu 1730 1735 1740 Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met 1745 1750 1755 1760 Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1765 1770 1775 Leu Ile Ser Tyr Lys Glu Asp Gln Arg Gly Glu Glu Pro Arg Arg Asn 1780 1785 1790 Phe Val Lys Pro Asn Glu Thr Lys Ile Tyr Phe Trp Lys Val Gln His 1795 1800 1805 His Met Ala Pro Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr 1810 1815 1820 Phe Ser Asp Val Asp Leu Glu Arg Asp Met His Ser Gly Leu Ile Gly 1825 1830 1835 1840 Pro Leu Leu Ile Cys His Ala Asn Thr Leu Asn Pro Ala His Gly Arg 1845 1850 1855 Gln Val Ser Val Gln Glu Phe Ala Leu Leu Phe Thr Ile Phe Asp Glu 1860 1865 1870 Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val Lys Arg Asn Cys Lys Thr 1875 1880 1885 Pro Cys Asn Phe Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg 1890 1895 1900 Phe His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val 1905 1910 1915 1920 Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Asn 1925 1930 1935 Asn Glu Asn Ile Gln Ser Ile His Phe Ser Gly His Val Phe Thr Val 1940 1945 1950 Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly 1955 1960 1965 Val Phe Glu Thr Leu Glu Met Ile Pro Ser Arg Ala Gly Ile Trp Arg 1970 1975 1980 Val Glu Cys Leu Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Leu 1985 1990 1995 2000 Phe Leu Val Tyr Ser Lys Gln Cys Gln Ile Pro Leu Gly Met Ala Ser 2005 2010 2015 Gly Ser Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly His Tyr Gly Gln 2020 2025 2030 Trp Ala Pro Asn Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala 2035 2040 2045 Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala 2050 2055 2060 Pro Met Ile Val His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 2065 2070 2075 2080 Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly 2085 2090 2095 Lys Lys Trp Leu Ser Tyr Gln Gly Asn Ser Thr Gly Thr Leu Met Val 2100 2105 2110 Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ser Phe Asn 2115 2120 2125 Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Ser Ser 2130 2135 2140 Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser 2145 2150 2155 2160 Cys Ser Ile Pro Leu Gly Met Glu Ser Lys Val Ile Ser Asp Thr Gln 2165 2170 2175 Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro 2180 2185 2190 Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro 2195 2200 2205 Gln Val Asn Asp Pro Lys Gln Trp Leu Gln Val Asp Leu Gln Lys Thr 2210 2215 2220 Met Lys Val Thr Gly Ile Ile Thr Gln Gly Val Lys Ser Leu Phe Thr 2225 2230 2235 2240 Ser Met Phe Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His 2245 2250 2255 His Trp Thr Gln Ile Leu Tyr Asn Gly Lys Val Lys Val Phe Gln Gly 2260 2265 2270 Asn Gln Asp Ser Ser Thr Pro Met Met Asn Ser Leu Asp Pro Pro Leu 2275 2280 2285 Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ile Trp Glu His Gln Ile 2290 2295 2300 Ala Leu Arg Leu Glu Ile Leu Gly Cys Glu Ala Gln Gln Gln Tyr 2305 2310 2315 9 14 PRT Homo sapeins DOMAIN (1)...(14) Linker sequence 9 Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 1 5 10 10 4 PRT Homo sapiens 10 Arg His Gln Arg 1 11 24 PRT Sus Scrofa DOMAIN (1)...(24) Linker sequence 11 Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Pro 1 5 10 15 Pro Val Leu Arg Arg His Gln Arg 20 12 4371 DNA Homo sapiens CDS (1)...(4371) B domain-deleted factor VIII (HSQ) 12 atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc ctt ttg cga ttc 48 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 tgc ttt agt gcc acc aga aga tac tac ctg ggt gca gtg gaa ctg tca 96 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tat atg caa agt gat ctc ggt gag ctg cct gtg gac gca aga 144 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45 ttt cct cct aga gtg cca aaa tct ttt cca ttc aac acc tca gtc gtg 192 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60 tac aaa aag act ctg ttt gta gaa ttc acg gtt cac ctt ttc aac atc 240 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile 65 70 75 80 gct aag cca agg cca ccc tgg atg ggt ctg cta ggt cct acc atc cag 288 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 gct gag gtt tat gat aca gtg gtc att aca ctt aag aac atg gct tcc 336 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 cat cct gtc agt ctt cat gct gtt ggt gta tcc tac tgg aaa gct tct 384 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 gag gga gct gaa tat gat gat cag acc agt caa agg gag aaa gaa gat 432 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140 gat aaa gtc ttc cct ggt gga agc cat aca tat gtc tgg cag gtc ctg 480 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 aaa gag aat ggt cca atg gcc tct gac cca ctg tgc ctt acc tac tca 528 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 tat ctt tct cat gtg gac ctg gta aaa gac ttg aat tca ggc ctc att 576 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc aag gaa aag aca 624 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 cag acc ttg cac aaa ttt ata cta ctt ttt gct gta ttt gat gaa ggg 672 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 aaa agt tgg cac tca gaa aca aag aac tcc ttg atg cag gat agg gat 720 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 gct gca tct gct cgg gcc tgg cct aaa atg cac aca gtc aat ggt tat 768 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 gta aac agg tct ctg cca ggt ctg att gga tgc cac agg aaa tca gtc 816 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270 tat tgg cat gtg att gga atg ggc acc act cct gaa gtg cac tca ata 864 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285 ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat cgc cag gcg tcc 912 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300 ttg gaa atc tcg cca ata act ttc ctt act gct caa aca ctc ttg atg 960 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 gac ctt gga cag ttt cta ctg ttt tgt cat atc tct tcc cac caa cat 1008 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 gat ggc atg gaa gct tat gtc aaa gta gac agc tgt cca gag gaa ccc 1056 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 caa cta cga atg aaa aat aat gaa gaa gcg gaa gac tat gat gat gat 1104 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365 ctt act gat tct gaa atg gat gtg gtc agg ttt gat gat gac aac tct 1152 Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380 cct tcc ttt atc caa att cgc tca gtt gcc aag aag cat cct aaa act 1200 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gta cat tac att gct gct gaa gag gag gac tgg gac tat gct ccc 1248 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 tta gtc ctc gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac 1296 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 aat ggc cct cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg 1344 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 gca tac aca gat gaa acc ttt aag acg cgt gaa gct att cag cat gaa 1392 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 tca gga atc ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat caa gca agc aga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cac gga atc act gat gtc cgt cct ttg tat tca agg aga tta cca aaa 1536 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 ggt gta aaa cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc 1584 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 aaa tat aaa tgg aca gtg act gta gaa gat ggg cca act aaa tca gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 ccg cgg tgc ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 gat cta gct tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gat caa aga gga aac cag ata atg tca gac aag agg aat gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttt gat gag aac cga agc tgg tac ctc aca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 aat ata caa cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 cca gag ttc caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agc att gga gca cag act gac ttc ctt tct gtc ttc ttc 2016 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct gga tat acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 cta ttc cca ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt cta tgg att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc 2160 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 atg acc gcc tta ctg aag gtt tct agt tgt gac aag aac act ggt gat 2208 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 tat tac gag gac agt tat gaa gat att tca gca tac ttg ctg agt aaa 2256 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 aac aat gcc att gaa cct agg agc ttc tct cag aat cca cca gtc ttg 2304 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765 aaa cgc cat caa cgg gaa ata act cgt act act ctt cag tca gat caa 2352 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780 gag gaa att gac tat gat gat acc ata tca gtt gaa atg aag aag gaa 2400 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 gat ttt gac att tat gat gag gat gaa aat cag agc ccc cgc agc ttt 2448 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815 caa aag aaa aca cga cac tat ttt att gct gca gtg gag agg ctc tgg 2496 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830 gat tat ggg atg agt agc tcc cca cat gtt cta aga aac agg gct cag 2544 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 agt ggc agt gtc cct cag ttc aag aaa gtt gtt ttc cag gaa ttt act 2592 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 gat ggc tcc ttt act cag ccc tta tac cgt gga gaa cta aat gaa cat 2640 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 ttg gga ctc ctg ggg cca tat ata aga gca gaa gtt gaa gat aat atc 2688 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895 atg gta act ttc aga aat cag gcc tct cgt ccc tat tcc ttc tat tct 2736 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910 agc ctt att tct tat gag gaa gat cag agg caa gga gca gaa cct aga 2784 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925 aaa aac ttt gtc aag cct aat gaa acc aaa act tac ttt tgg aaa gtg 2832 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 caa cat cat atg gca ccc act aaa gat gag ttt gac tgc aaa gcc tgg 2880 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 gct tat ttc tct gat gtt gac ctg gaa aaa gat gtg cac tca ggc ctg 2928 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 att gga ccc ctt ctg gtc tgc cac act aac aca ctg aac cct gct cat 2976 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 ggg aga caa gtg aca gta cag gaa ttt gct ctg ttt ttc acc atc ttt 3024 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005 gat gag acc aaa agc tgg tac ttc act gaa aat atg gaa aga aac tgc 3072 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys 1010 1015 1020 agg gct ccc tgc aat atc cag atg gaa gat ccc act ttt aaa gag aat 3120 Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 1025 1030 1035 1040 tat cgc ttc cat gca atc aat ggc tac ata atg gat aca cta cct ggc 3168 Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1045 1050 1055 tta gta atg gct cag gat caa agg att cga tgg tat ctg ctc agc atg 3216 Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1060 1065 1070 ggc agc aat gaa aac atc cat tct att cat ttc agt gga cat gtg ttc 3264 Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1075 1080 1085 act gta cga aaa aaa gag gag tat aaa atg gca ctg tac aat ctc tat 3312 Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1090 1095 1100 cca ggt gtt ttt gag aca gtg gaa atg tta cca tcc aaa gct gga att 3360 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 1105 1110 1115 1120 tgg cgg gtg gaa tgc ctt att ggc gag cat cta cat gct ggg atg agc 3408 Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser 1125 1130 1135 aca ctt ttt ctg gtg tac agc aat aag tgt cag act ccc ctg gga atg 3456 Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 1140 1145 1150 gct tct gga cac att aga gat ttt cag att aca gct tca gga caa tat 3504 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1155 1160 1165 gga cag tgg gcc cca aag ctg gcc aga ctt cat tat tcc gga tca atc 3552 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 1170 1175 1180 aat gcc tgg agc acc aag gag ccc ttt tct tgg atc aag gtg gat ctg 3600 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1185 1190 1195 1200 ttg gca cca atg att att cac ggc atc aag acc cag ggt gcc cgt cag 3648 Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 1205 1210 1215 aag ttc tcc agc ctc tac atc tct cag ttt atc atc atg tat agt ctt 3696 Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 1220 1225 1230 gat ggg aag aag tgg cag act tat cga gga aat tcc act gga acc tta 3744 Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245 atg gtc ttc ttt ggc aat gtg gat tca tct ggg ata aaa cac aat att 3792 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile 1250 1255 1260 ttt aac cct cca att att gct cga tac atc cgt ttg cac cca act cat 3840 Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 1265 1270 1275 1280 tat agc att cgc agc act ctt cgc atg gag ttg atg ggc tgt gat tta 3888 Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1285 1290 1295 aat agt tgc agc atg cca ttg gga atg gag agt aaa gca ata tca gat 3936 Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1300 1305 1310 gca cag att act gct tca tcc tac ttt acc aat atg ttt gcc acc tgg 3984 Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1315 1320 1325 tct cct tca aaa gct cga ctt cac ctc caa ggg agg agt aat gcc tgg 4032 Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1330 1335 1340 aga cct cag gtg aat aat cca aaa gag tgg ctg caa gtg gac ttc cag 4080 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 1345 1350 1355 1360 aag aca atg aaa gtc aca gga gta act act cag gga gta aaa tct ctg 4128 Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu 1365 1370 1375 ctt acc agc atg tat gtg aag gag ttc ctc atc tcc agc agt caa gat 4176 Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 1380 1385 1390 ggc cat cag tgg act ctc ttt ttt cag aat ggc aaa gta aag gtt ttt 4224 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1395 1400 1405 cag gga aat caa gac tcc ttc aca cct gtg gtg aac tct cta gac cca 4272 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 1410 1415 1420 ccg tta ctg act cgc tac ctt cga att cac ccc cag agt tgg gtg cac 4320 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1425 1430 1435 1440 cag att gcc ctg agg atg gag gtt ctg ggc tgc gag gca cag gac ctc 4368 Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 1445 1450 1455 tac 4371 Tyr 13 1457 PRT Homo sapiens 13 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365 Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys 1010 1015 1020 Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 1025 1030 1035 1040 Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1045 1050 1055 Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1060 1065 1070 Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1075 1080 1085 Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1090 1095 1100 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 1105 1110 1115 1120 Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser 1125 1130 1135 Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 1140 1145 1150 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1155 1160 1165 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 1170 1175 1180 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1185 1190 1195 1200 Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 1205 1210 1215 Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 1220 1225 1230 Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile 1250 1255 1260 Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 1265 1270 1275 1280 Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1285 1290 1295 Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1300 1305 1310 Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1315 1320 1325 Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1330 1335 1340 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 1345 1350 1355 1360 Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu 1365 1370 1375 Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 1380 1385 1390 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1395 1400 1405 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 1410 1415 1420 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1425 1430 1435 1440 Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 1445 1450 1455 Tyr 14 4401 DNA Artificial Sequence hybrid human and porcine factor VIII sequence 14 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt ctc tgg gtc ctt ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 aag aat gtc att gaa cct agg agc ttt gcc cag aat tca aga ccc cct 2304 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 agt gcg agc gct cca aag cct ccg gtc ctg cga cgg cat cag agg gac 2352 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 ata agc ctt cct act ttt cag ccg gag gaa gac aaa atg gac tat gat 2400 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 gat atc ttc tca act gaa acg aag gga gaa gat ttt gac att tac ggt 2448 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 gag gat gaa aat cag gac cct cgc agc ttt cag aag aga acc cga cac 2496 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 gat gat cag gag caa ggg gca gaa cct cga cac aac ttc gtc cag cca 2832 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 aat gaa acc aga act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 gtg gaa atg cta ccg tcc aaa gtt gga att tgg cga ata gaa tgc ctg 3408 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 agc aag aag tgt cag act ccc ctg gga atg gct tct gga cac att aga 3504 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 gat ttt cag att aca gct tca gga caa tat gga cag tgg gcc cca aag 3552 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 gag ccc ttt tct tgg atc aag gtg gat ctg ttg gca cca atg att att 3648 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 cac ggc atc aag acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac 3696 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 atc tct cag ttt atc atc atg tat agt ctt gat ggg aag aag tgg cag 3744 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 act tat cga gga aat tcc act gga acc tta atg gtc ttc ttt ggc aat 3792 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 gtg gat tca tct ggg ata aaa cac aat att ttt aac cct cca att att 3840 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 gct cga tac atc cgt ttg cac cca act cat tat agc att cgc agc act 3888 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 ctt cgc atg gag ttg atg ggc tgt gat tta aat agt tgc agc atg cca 3936 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 ttg gga atg gag agt aaa gca ata tca gat gca cag att act gct tca 3984 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 tcc tac ttt acc aat atg ttt gcc acc tgg tct cct tca aaa gct cga 4032 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 ctt cac ctc caa ggg agg agt aat gcc tgg aga cct cag gtg aat aat 4080 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc aca 4128 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 gga gta act act cag gga gta aaa tct ctg ctt acc agc atg tat gtg 4176 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 aag gag ttc ctc atc tcc agc agt caa gat ggc cat cag tgg act ctc 4224 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac tcc 4272 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 ttc aca cct gtg gtg aac tct cta gac cca ccg tta ctg act cgc tac 4320 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 ctt cga att cac ccc cag agt tgg gtg cac cag att gcc ctg agg atg 4368 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 gag gtt ctg ggc tgc gag gca cag gac ctc tac 4401 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 15 1467 PRT Artificial Sequence HP44/OL-- factor VIII having the following domains A1p-A2p-app-A3p-C1h-C2h 15 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430 Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445 Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510 Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735 Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 16 4371 DNA Artificial Sequence hybrid human and porcine factor VIII sequence 16 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tca gtt gcc aag aag cat cct aaa act 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gta cat tac att gct gct gaa gag gag gac tgg gac tat gct ccc 1248 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 tta gtc ctc gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac 1296 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 aat ggc cct cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg 1344 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 gca tac aca gat gaa acc ttt aag acg cgt gaa gct att cag cat gaa 1392 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 tca gga atc ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat caa gca agc aga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cac gga atc act gat gtc cgt cct ttg tat tca agg aga tta cca aaa 1536 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 ggt gta aaa cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc 1584 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 aaa tat aaa tgg aca gtg act gta gaa gat ggg cca act aaa tca gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 ccg cgg tgc ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 gat cta gct tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gat caa aga gga aac cag ata atg tca gac aag agg aat gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttt gat gag aac cga agc tgg tac ctc aca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 aat ata caa cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 cca gag ttc caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agc att gga gca cag act gac ttc ctt tct gtc ttc ttc 2016 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct gga tat acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 cta ttc cca ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt cta tgg att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc 2160 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 atg acc gcc tta ctg aag gtt tct agt tgt gac aag aac act ggt gat 2208 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 tat tac gag gac agt tat gaa gat att tca gca tac ttg ctg agt aaa 2256 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 aac aat gcc att gaa cct agg agc ttc tct cag aat cca cca gtc ttg 2304 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765 aaa cgc cat caa cgg gaa ata act cgt act act ctt cag tca gat caa 2352 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780 gag gaa att gac tat gat gat acc ata tca gtt gaa atg aag aag gaa 2400 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 gat ttt gac att tat gat gag gat gaa aat cag agc ccc cgc agc ttt 2448 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815 caa aag aaa aca cga cac tat ttt att gct gca gtg gag agg ctc tgg 2496 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830 gat tat ggg atg agt agc tcc cca cat gtt cta aga aac agg gct cag 2544 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 agt ggc agt gtc cct cag ttc aag aaa gtt gtt ttc cag gaa ttt act 2592 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 gat ggc tcc ttt act cag ccc tta tac cgt gga gaa cta aat gaa cat 2640 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 ttg gga ctc ctg ggg cca tat ata aga gca gaa gtt gaa gat aat atc 2688 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895 atg gta act ttc aga aat cag gcc tct cgt ccc tat tcc ttc tat tct 2736 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910 agc ctt att tct tat gag gaa gat cag agg caa gga gca gaa cct aga 2784 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925 aaa aac ttt gtc aag cct aat gaa acc aaa act tac ttt tgg aaa gtg 2832 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 caa cat cat atg gca ccc act aaa gat gag ttt gac tgc aaa gcc tgg 2880 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 gct tat ttc tct gat gtt gac ctg gaa aaa gat gtg cac tca ggc ctg 2928 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 att gga ccc ctt ctg gtc tgc cac act aac aca ctg aac cct gct cat 2976 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 ggg aga caa gtg aca gta cag gaa ttt gct ctg ttt ttc acc atc ttt 3024 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005 gat gag acc aaa agc tgg tac ttc act gaa aat atg gaa aga aac tgc 3072 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys 1010 1015 1020 agg gct ccc tgc aat atc cag atg gaa gat ccc act ttt aaa gag aat 3120 Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 1025 1030 1035 1040 tat cgc ttc cat gca atc aat ggc tac ata atg gat aca cta cct ggc 3168 Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1045 1050 1055 tta gta atg gct cag gat caa agg att cga tgg tat ctg ctc agc atg 3216 Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1060 1065 1070 ggc agc aat gaa aac atc cat tct att cat ttc agt gga cat gtg ttc 3264 Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1075 1080 1085 act gta cga aaa aaa gag gag tat aaa atg gca ctg tac aat ctc tat 3312 Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1090 1095 1100 cca ggt gtt ttt gag aca gtg gaa atg tta cca tcc aaa gct gga att 3360 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 1105 1110 1115 1120 tgg cgg gtg gaa tgc ctt att ggc gag cat cta cat gct ggg atg agc 3408 Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser 1125 1130 1135 aca ctt ttt ctg gtg tac agc aat aag tgt cag act ccc ctg gga atg 3456 Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 1140 1145 1150 gct tct gga cac att aga gat ttt cag att aca gct tca gga caa tat 3504 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1155 1160 1165 gga cag tgg gcc cca aag ctg gcc aga ctt cat tat tcc gga tca atc 3552 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 1170 1175 1180 aat gcc tgg agc acc aag gag ccc ttt tct tgg atc aag gtg gat ctg 3600 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1185 1190 1195 1200 ttg gca cca atg att att cac ggc atc aag acc cag ggt gcc cgt cag 3648 Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 1205 1210 1215 aag ttc tcc agc ctc tac atc tct cag ttt atc atc atg tat agt ctt 3696 Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 1220 1225 1230 gat ggg aag aag tgg cag act tat cga gga aat tcc act gga acc tta 3744 Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245 atg gtc ttc ttt ggc aat gtg gat tca tct ggg ata aaa cac aat att 3792 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile 1250 1255 1260 ttt aac cct cca att att gct cga tac atc cgt ttg cac cca act cat 3840 Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 1265 1270 1275 1280 tat agc att cgc agc act ctt cgc atg gag ttg atg ggc tgt gat tta 3888 Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1285 1290 1295 aat agt tgc agc atg cca ttg gga atg gag agt aaa gca ata tca gat 3936 Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1300 1305 1310 gca cag att act gct tca tcc tac ttt acc aat atg ttt gcc acc tgg 3984 Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1315 1320 1325 tct cct tca aaa gct cga ctt cac ctc caa ggg agg agt aat gcc tgg 4032 Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1330 1335 1340 aga cct cag gtg aat aat cca aaa gag tgg ctg caa gtg gac ttc cag 4080 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 1345 1350 1355 1360 aag aca atg aaa gtc aca gga gta act act cag gga gta aaa tct ctg 4128 Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu 1365 1370 1375 ctt acc agc atg tat gtg aag gag ttc ctc atc tcc agc agt caa gat 4176 Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 1380 1385 1390 ggc cat cag tgg act ctc ttt ttt cag aat ggc aaa gta aag gtt ttt 4224 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1395 1400 1405 cag gga aat caa gac tcc ttc aca cct gtg gtg aac tct cta gac cca 4272 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 1410 1415 1420 ccg tta ctg act cgc tac ctt cga att cac ccc cag agt tgg gtg cac 4320 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1425 1430 1435 1440 cag att gcc ctg agg atg gag gtt ctg ggc tgc gag gca cag gac ctc 4368 Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 1445 1450 1455 tac 4371 Tyr 17 1457 PRT Artificial Sequence HP46/SQ-- factor VIII having the following domains A1p-A2h-aph-A3h-C1h-C2h 17 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys 1010 1015 1020 Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 1025 1030 1035 1040 Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1045 1050 1055 Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1060 1065 1070 Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1075 1080 1085 Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1090 1095 1100 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 1105 1110 1115 1120 Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser 1125 1130 1135 Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 1140 1145 1150 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1155 1160 1165 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile 1170 1175 1180 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1185 1190 1195 1200 Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 1205 1210 1215 Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu 1220 1225 1230 Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile 1250 1255 1260 Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 1265 1270 1275 1280 Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1285 1290 1295 Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1300 1305 1310 Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1315 1320 1325 Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1330 1335 1340 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 1345 1350 1355 1360 Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu 1365 1370 1375 Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 1380 1385 1390 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1395 1400 1405 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro 1410 1415 1420 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1425 1430 1435 1440 Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 1445 1450 1455 Tyr 18 4401 DNA Artificial Sequence hybrid human and porcine factor VIII sequence 18 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tca gtt gcc aag aag cat cct aaa act 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gta cat tac att gct gct gaa gag gag gac tgg gac tat gct ccc 1248 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 tta gtc ctc gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac 1296 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 aat ggc cct cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg 1344 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 gca tac aca gat gaa acc ttt aag acg cgt gaa gct att cag cat gaa 1392 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 tca gga atc ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat caa gca agc aga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cac gga atc act gat gtc cgt cct ttg tat tca agg aga tta cca aaa 1536 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 ggt gta aaa cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc 1584 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 aaa tat aaa tgg aca gtg act gta gaa gat ggg cca act aaa tca gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 ccg cgg tgc ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 gat cta gct tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gat caa aga gga aac cag ata atg tca gac aag agg aat gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttt gat gag aac cga agc tgg tac ctc aca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 aat ata caa cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 cca gag ttc caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agc att gga gca cag act gac ttc ctt tct gtc ttc ttc 2016 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct gga tat acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 cta ttc cca ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt cta tgg att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc 2160 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 atg acc gcc tta ctg aag gtt tct agt tgt gac aag aac act ggt gat 2208 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 tat tac gag gac agt tat gaa gat att tca gca tac ttg ctg agt aaa 2256 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 aac aat gcc att gaa cct agg agc ttt gcc cag aat tca aga ccc cct 2304 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 agt gcg agc gct cca aag cct ccg gtc ctg cga cgg cat cag agg gac 2352 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 ata agc ctt cct act ttt cag ccg gag gaa gac aaa atg gac tat gat 2400 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 gat atc ttc tca act gaa acg aag gga gaa gat ttt gac att tac ggt 2448 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 gag gat gaa aat cag gac cct cgc agc ttt cag aag aga acc cga cac 2496 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 gat gat cag gag caa ggg gca gaa cct cga cac aac ttc gtc cag cca 2832 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 aat gaa acc aga act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 gtg gaa atg cta ccg tcc aaa gtt gga att tgg cga ata gaa tgc ctg 3408 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 agc aag aag tgt cag act ccc ctg gga atg gct tct gga cac att aga 3504 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 gat ttt cag att aca gct tca gga caa tat gga cag tgg gcc cca aag 3552 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 gag ccc ttt tct tgg atc aag gtg gat ctg ttg gca cca atg att att 3648 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 cac ggc atc aag acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac 3696 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 atc tct cag ttt atc atc atg tat agt ctt gat ggg aag aag tgg cag 3744 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 act tat cga gga aat tcc act gga acc tta atg gtc ttc ttt ggc aat 3792 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 gtg gat tca tct ggg ata aaa cac aat att ttt aac cct cca att att 3840 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 gct cga tac atc cgt ttg cac cca act cat tat agc att cgc agc act 3888 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 ctt cgc atg gag ttg atg ggc tgt gat tta aat agt tgc agc atg cca 3936 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 ttg gga atg gag agt aaa gca ata tca gat gca cag att act gct tca 3984 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 tcc tac ttt acc aat atg ttt gcc acc tgg tct cct tca aaa gct cga 4032 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 ctt cac ctc caa ggg agg agt aat gcc tgg aga cct cag gtg aat aat 4080 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc aca 4128 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 gga gta act act cag gga gta aaa tct ctg ctt acc agc atg tat gtg 4176 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 aag gag ttc ctc atc tcc agc agt caa gat ggc cat cag tgg act ctc 4224 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac tcc 4272 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 ttc aca cct gtg gtg aac tct cta gac cca ccg tta ctg act cgc tac 4320 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 ctt cga att cac ccc cag agt tgg gtg cac cag att gcc ctg agg atg 4368 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 gag gtt ctg ggc tgc gag gca cag gac ctc tac 4401 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 19 1467 PRT Artificial Sequence HP47/OL -- factor VIII having the following domains A1p-A2h-app-A3p-C1h-C2h 19 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765 Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780 Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp 785 790 795 800 Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815 Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940 Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu 1125 1130 1135 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 20 4401 DNA Artificial Sequence hybrid human and porcine factor VIII sequence 20 atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 tct ccc ttt atc caa atc cgc tca gtt gcc aag aag cat cct aaa act 1200 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 tgg gta cat tac att gct gct gaa gag gag gac tgg gac tat gct ccc 1248 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 tta gtc ctc gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac 1296 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 aat ggc cct cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg 1344 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 gca tac aca gat gaa acc ttt aag act cgt gaa gct att cag cat gaa 1392 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 tca gga atc ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg 1440 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 ttg att ata ttt aag aat caa gca agc aga cca tat aac atc tac cct 1488 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 cac gga atc act gat gtc cgt cct ttg tat tca agg aga tta cca aaa 1536 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 ggt gta aaa cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc 1584 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 aaa tat aaa tgg aca gtg act gta gaa gat ggg cca act aaa tca gat 1632 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga 1680 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 gat cta gct tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 tct gta gat caa aga gga aac cag ata atg tca gac aag agg aat gtc 1776 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 atc ctg ttt tct gta ttt gat gag aac cga agc tgg tac ctc aca gag 1824 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 aat ata caa cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat 1872 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 cca gag ttc caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt 1920 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg 1968 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 tac att cta agc att gga gca cag act gac ttc ctt tct gtc ttc ttc 2016 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 tct gga tat acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 cta ttc cca ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca 2112 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 ggt cta tgg att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc 2160 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 atg acc gcc tta ctg aag gtt tct agt tgt gac aag aac act ggt gat 2208 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 tat tac gag gac agt tat gaa gat att tca gca tac ttg ctg agt aaa 2256 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 aac aat gcc att gaa cct agg agc ttc tcc cag aat tca aga cac cct 2304 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765 agc act agg tct caa aac cca cca gtc ttg aaa cgc cat caa cgg gaa 2352 Ser Thr Arg Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 770 775 780 ata act cgt act act ctt cag tca gat caa gag gaa att gac tat gat 2400 Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp 785 790 795 800 gat acc ata tca gtt gaa atg aag aag gaa gat ttt gac att tat gat 2448 Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp 805 810 815 gag gat gaa aat cag agc ccc cgc agc ttt caa aag aga acc cga cac 2496 Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 gat gat cag gag caa ggg gca gaa cct cga aaa aac ttt gtc aag cct 2832 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro 930 935 940 aat gaa acc aaa act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880 Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 gtg gaa atg cta ccg tcc aaa gtt gga att tgg cgg aat aga tgc ctg 3408 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Asn Arg Cys Leu 1125 1130 1135 att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 agc aag aag tgt cag act ccc ctg gga atg gct tct gga cac att aga 3504 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 gat ttt cag att aca gct tca gga caa tat gga cag tgg gcc cca aag 3552 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 gag ccc ttt tct tgg atc aag gtg gat ctg ttg gca cca atg att att 3648 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 cac ggc atc aag acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac 3696 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 atc tct cag ttt atc atc atg tat agt ctt gat ggg aag aag tgg cag 3744 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 act tat cga gga aat tcc act gga acc tta atg gtc ttc ttt ggc aat 3792 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 gtg gat tca tct ggg ata aaa cac aat att ttt aac cct cca att att 3840 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 gct cga tac atc cgt ttg cac cca act cat tat agc att cgc agc act 3888 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 ctt cgc atg gag ttg atg ggc tgt gat tta aat agt tgc agc atg cca 3936 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 ttg gga atg gag agt aaa gca ata tca gat gca cag att act gct tca 3984 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 tcc tac ttt acc aat atg ttt gcc acc tgg tct cct tca aaa gct cga 4032 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 ctt cac ctc caa ggg agg agt aat gcc tgg aga cct cag gtg aat aat 4080 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc aca 4128 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 gga gta act act cag gga gta aaa tct ctg ctt acc agc atg tat gtg 4176 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 aag gag ttc ctc atc tcc agc agt caa gat ggc cat cag tgg act ctc 4224 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac tcc 4272 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 ttc aca cct gtg gtg aac tct cta gac cca ccg tta ctg act cgc tac 4320 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 ctt cga att cac ccc cag agt tgg gtg cac cag att gcc ctg agg atg 4368 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 gag gtt ctg ggc tgc gag gca cag gac ctc tac 4401 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 21 1467 PRT Artificial Sequence HP63/OL -- factor VIII variant 21 Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu 1 5 10 15 Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30 Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45 Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60 Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser 65 70 75 80 Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95 Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110 Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125 Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140 Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val 145 150 155 160 Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175 Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190 Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205 Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220 Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met 225 230 235 240 Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255 Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270 Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285 Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300 Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu 305 310 315 320 Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335 His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350 Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365 Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380 Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765 Ser Thr Arg Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 770 775 780 Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp 785 790 795 800 Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp 805 810 815 Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830 Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845 Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860 Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln 865 870 875 880 Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895 Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910 Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925 Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro 930 935 940 Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 945 950 955 960 Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975 Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990 Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005 Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1010 1015 1020 Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu 1025 1030 1035 1040 Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile 1045 1050 1055 Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn 1060 1065 1070 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1075 1080 1085 His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu 1090 1095 1100 Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1105 1110 1115 1120 Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Asn Arg Cys Leu 1125 1130 1135 Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr 1140 1145 1150 Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1155 1160 1165 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys 1170 1175 1180 Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys 1185 1190 1195 1200 Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1205 1210 1215 His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1220 1225 1230 Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245 Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1250 1255 1260 Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile 1265 1270 1275 1280 Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr 1285 1290 1295 Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1300 1305 1310 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1315 1320 1325 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg 1330 1335 1340 Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1345 1350 1355 1360 Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1365 1370 1375 Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val 1380 1385 1390 Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1395 1400 1405 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser 1410 1415 1420 Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr 1425 1430 1435 1440 Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1445 1450 1455 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465 US 20120270267 A1 20121025 US 13516780 20110104 13 EP 10150062.7 20100104 EP 10150063.5 20100104 20060101 A
C
12 N 9 26 F I 20121025 US B H
20060101 A
C
12 N 9 16 L I 20121025 US B H
20060101 A
C
12 P 19 14 L I 20121025 US B H
20060101 A
C
12 P 7 14 L I 20121025 US B H
20060101 A
C
12 P 7 16 L I 20121025 US B H
20060101 A
C
12 P 7 46 L I 20121025 US B H
20060101 A
C
12 P 7 56 L I 20121025 US B H
20060101 A
C
12 P 7 20 L I 20121025 US B H
20060101 A
C
12 P 7 60 L I 20121025 US B H
20060101 A
C
12 P 13 08 L I 20121025 US B H
20060101 A
C
12 P 21 04 L I 20121025 US B H
20060101 A
C
12 N 15 63 L I 20121025 US B H
20060101 A
C
12 N 1 15 L I 20121025 US B H
20060101 A
C
12 N 1 19 L I 20121025 US B H
20060101 A
C
12 N 5 10 L I 20121025 US B H
20060101 A
C
12 N 1 21 L I 20121025 US B H
20060101 A
C
11 D 3 386 L I 20121025 US B H
20060101 A
C
12 N 15 11 L I 20121025 US B H
20060101 A
C
12 N 15 56 L I 20121025 US B H
20060101 A
C
12 N 9 20 L I 20121025 US B H
US 435 696 435201 435198 435196 435 99 435162 435160 435145 435139 435159 435138 435115 4353201 43525231 43525235 43525233 43525234 435325 435348 43525411 4352542 43525422 43525421 43525423 4352543 4352547 4352548 4352544 4352545 4352546 435412 435417 435414 435415 435419 4352523 510392 536 232 Alpha-Amylases US 61304092 20100212 US 61333930 20100512 US 61354775 20100615 US 61354817 20100615 US 61355230 20100616 US 61362536 20100708 Andersen Carsten
Vaerloese DK
omitted DK
Poulsen Thomas Agersten
Ballerup DK
omitted DK
NOVOZYMES A/S 03
Bagsvaerd DK
WO PCT/EP2011/050075 00 20110104 20120618

The present invention relates to alpha-amylase variants, polynucleotides encoding the variants and nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variant enzymes.

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REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to alpha-amylase variants, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.

2. Description of Related Art

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.

Alpha-amylases are used commercially for a variety of purposes such as in the initial stages of starch processing (e.g., liquefaction); in wet milling processes; and in alcohol production from carbohydrate sources. They are also used as cleaning agents or adjuncts in detergent matrices; in the textile industry for starch desizing; in baking applications; in the beverage industry; in oil fields in drilling processes; in recycling processes, e.g., for de-inking paper; and in animal feed.

One of the first bacterial alpha-amylases to be used was an alpha-amylase from B. licheniformis, also known as Termamyl™, which has been extensively characterized and the crystal structure has been determined for this enzyme. Alkaline amylases, such as the alpha-amylase derived from Bacillus sp. strains NCIB 12289, NCIB 12512, NCIB 12513, and DSM 9375 (disclosed in WO 95/26397), form a particular group of alpha-amylases that are useful in detergents. Many of these known bacterial amylases have been modified in order to improve their functionality in a particular application.

Termamyl™ and many highly efficient alpha-amylases require calcium for activity. The crystal structure of Termamyl™ shows that three calcium atoms are bound to the alpha-amylase structure coordinated by negatively charged amino acid residues. This requirement for calcium is a disadvantage in applications where strong chelating compounds are present, such as in detergents or during ethanol production from whole grains, where the plant material comprises a large amount of natural chelators such as phytate.

Calcium-insensitive amylases are known, e.g., the alpha-amylases disclosed in EP 1022334 and WO 03/083054, and a Bacillus circulans alpha-amylase having the sequence disclosed in UNIPROT:Q03657.

It would therefore be beneficial to provide alpha-amylases with reduced calcium sensitivity.

SUMMARY OF THE INVENTION

The present invention provides alpha-amylase variants comprising an A-domain of a calcium-sensitive alpha-amylase, a B-domain which has at least 55% and less than 100% sequence identity with the B-domain of SEQ ID NO: 13, and a C-domain of a calcium-sensitive alpha-amylase.

The present invention also relates to isolated polynucleotides encoding an alpha-amylase variant, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of producing a variant of a parent alpha-amylase.

The present invention also relates to the use of the variants in starch processing (e.g., liquefaction); wet milling processes; alcohol production from carbohydrate sources; detergents; dishwashing compositions; starch desizing in the textile industry; baking applications; the beverage industry; oil fields in drilling processes; recycling processes, e.g., for de-inking paper, and animal feed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of SEQ ID NOS: 1-16, 29 and 30.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides alpha-amylase variants comprising an A-domain of a calcium-sensitive alpha-amylase, a B-domain which has at least 50% and less than 100% sequence identity with the B-domain of SEQ ID NO: 13, and a C-domain of a calcium-sensitive alpha-amylase.

DEFINITIONS

A, B and C-Domains: The structure of alpha-amylases comprises three distinct domains A, B and C, see, e.g., Machius et al., 1995, J. Mol. Biol. 246: 545-559. The term “domain” means a region of a polypeptide that in itself forms a distinct and independent substructure of the whole molecule. Alpha-amylases consist of a beta/alpha-8 barrel harboring the active site, which is denoted the A-domain, a rather long loop between the beta-sheet 3 and alpha-helix 3, which is denoted the B-domain, and a C-domain and in some cases also a carbohydrate binding domain (e.g., WO 2005/001064; Machius et al., supra).

The domains of an alpha-amylase can be determined by structure analysis such as by using crystallographically techniques. An alternative method for determining the domains of an alpha-amylase is by sequence alignment of the amino acid sequence of the alpha-amylase with another alpha-amylase for which the domains have been determined. The sequence that aligns with, e.g., the B-domain sequence in the alpha-amylase for which the B-domain has been determined can be considered the B-domain for the given alpha-amylase.

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) are a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.

Calcium-insensitive amylase means an alpha-amylase that does not require the presence of calcium for optimal activity and/or for maintaining the active conformation/structure.

Calcium-sensitive amylase means an alpha-amylase that requires the presence of calcium to retain its structure and/or to have full enzymatic activity. For some calcium-sensitive amylases it has been shown that they contains a calcium atom coordinated to acidic amino acid residues in the active conformation. A large number of calcium-sensitive alpha-amylases are known and have been used industrially because of their beneficial properties. Calcium-sensitive alpha-amylases are generally sensitive towards conditions that leads to loss of the calcium atom coordinated in their structure such as detergent compositions and fuel mass.

Calcium sensitivity is determined by incubating an alpha-amylase in the presence of a strong chelator and analyzing the impact of this incubation on the activity or stability of the alpha-amylase. A calcium-sensitive alpha-amylase will be less stable in the presence of a chelator or lose a major part or all of its activity during incubation whereas a calcium-insensitive alpha-amylase will not lose all of its activity or will lose only a minor part of the activity during incubation. Chelator strength may be evaluated using methods known in the art such as the methods disclosed in Nielsen et al., 2003, Anal. Biochem. 314: 227-234; and Nagarajan and Paine, 1984, J. Am. Oil Chem. Soc. 61(9): 1475-1478. Examples of strong chelators that may be used for such an assay are EGTA (ethylene glycol tetraacetic acid), EDTA (ethylene diamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid), DTMPA (diethylene triamine-penta-methylene phosphonic acid) and HEDP (1-hydroxyethan-1,1-diylbis(phosphonic acid)). Other strong chelators may be used to determine the calcium sensitivity of an alpha-amylase.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of its polypeptide product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.

Control sequence: The term “control sequence” means all components necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Improved property: The term “improved property” means a characteristic associated with a variant that is improved compared to other alpha-amylases. Such improved properties include, but are not limited to, altered temperature-dependent activity profile, thermostability, pH activity, pH stability, substrate specificity, product specificity, and chemical stability.

Isolated variant: The terms “isolated” and “purified” mean a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated. For example, a variant may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE and a polynucleotide may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a nucleotide sequence that encodes a mature polypeptide having alpha-amylase activity.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

Parent: The term “parent” alpha-amylase means an alpha-amylase to which an alteration is made to produce a variant of the present invention. The parent may be a naturally occurring (wild-type) polypeptide, or a variant thereof, prepared by any suitable means. For instance, the parent polypeptide may be a variant of a naturally occurring polyptide which has a modified or altered amino acid sequence. A parent may also be an allelic variant.

Polypeptide fragment: The term “polypeptide fragment” means a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has alpha-amylase activity. In one aspect, a fragment contains at least 481 amino acid residues, e.g., at least 483, at least 486, and at least 493 amino acid residues.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:


(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:


(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide sequence having one or more (several) nucleotides deleted from the 5′ and/or 3′ end of a mature polypeptide coding sequence; wherein the subsequence encodes a polypeptide fragment having alpha-amylase activity.

Variant: The term “variant” means a polypeptide having alpha-amylase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, of one or more (several) amino acid residues at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-5 amino acids adjacent to and following an amino acid occupying a position.

Wild-Type: The term “wild-type” means an alpha-amylase expressed by a naturally occurring microorganism, such as a bacterial, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, unless otherwise indicated, the hybrid polypeptide disclosed in SEQ ID NO: 27 (which has the sequence of amino acids 1-104 of Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 4), followed by amino acids 103-208 of Bacillus circulans alpha-amylase (SEQ ID NO: 13), followed by amino acids 211-515 of Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 4)) is used to determine the corresponding amino acid residue in another alpha-amylase. The amino acid sequence of another alpha-amylase is aligned with the mature polypeptide disclosed in SEQ ID NO: 27, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 27 can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.

Identification of the corresponding amino acid residue in another alpha-amylase can be confirmed by an alignment of multiple polypeptide sequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23: 2947-2948).

When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: 27 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more (several) representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Eng. 11: 739-747), and implementations of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567). These structural alignments can be used to predict the structurally and functionally corresponding amino acid residues in proteins within the same structural superfamily. This information, along with information derived from homology modeling and profile searches, can be used to predict which residues to mutate when moving mutations of interest from one protein to a close or remote homolog.

In describing the alpha-amylase variants of the present invention, the nomenclature described below is adapted for ease of reference. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used: original amino acid, position, original amino acid, new inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. Multiple insertions of amino acids are designated [Original amino acid, position, original amino acid, new inserted amino acid #1, new inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.

Different Alterations.

Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “Arg170Tyr,Glu” represents a substitution of arginge with tyrosine or glutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates the following variants:

Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly, and Tyr167Ala+Arg170Ala. Domains Calcium-Sensitive Alpha-Amylases

Examples of calcium-sensitive alpha-amylases include the following alpha-amylases:

1. Bacillus amyloliquefaciens alpha-amylase having the amino acid sequence of SEQ ID NO: 1;
2. Bacillus flavothermus amylase, AMY1048 described in WO 2005/001064, having the amino acid sequence of SEQ ID NO: 2;
3. Bacillus licheniformis alpha-amylase having the amino acid sequence of SEQ ID NO: 3,
4. Bacillus stearothermophilus alpha-amylase having the amino acid sequence of SEQ ID NO: 4;
5. Alpha-amylase AA560 derived from Bacillus sp. DSM 12649 described in WO 00/60060, having the amino acid sequence of SEQ ID NO: 5;
6. Alpha-amylase derived from Bacillus sp. strain NCIB 12512 described in WO 95/26397, having the amino acid sequence of SEQ ID NO: 6;
7. Alpha-amylase derived from Bacillus sp. strain NCIB 12513 described in WO 95/26397, having the amino acid sequence of SEQ ID NO: 7;
8. Alpha-amylase SP707 described by Tsukamoto et al., 1988, Biochem. Biophys. Res. Comm. 151: 25-31, having the amino acid sequence of SEQ ID NO: 8;
9. Alpha-amylase TS-22 having the amino acid sequence of SEQ ID NO: 9;
10. Alpha-amylase TS-23 described in J. Appl. Microbiology, 1997, 82: 325-334 (SWALL:q59222), having the amino acid sequence of SEQ ID NO: 10;
11. Alpha-amylase derived from Bacillus sp. KSM-AP1378 (FERM BP-3048) described in WO 97/00324, having the amino acid sequence of SEQ ID NO: 11;
12. Alpha-amylase derived from Bacillus sp. A 7-7 described in WO 02/10356, having the amino acid sequence of SEQ ID NO: 12;
13. Alpha-amylase derived from Bacillus stearothermophilus (Spezyme Xtra), having the amino acid sequence of SEQ ID NO: 29.
14. Cytophaga alpha-amylase described in Jeang et al., 2002, Appl. Environ. Microbiol. 68:3651-3654, having the amino acid sequence of SEQ ID NO: 30;
as well as hybrids and variants of any of these calcium-sensitive alpha-amylases.

Other calcium-sensitive alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811) and the alpha-amylases disclosed in WO 91/00353 and WO 94/18314.

The calcium-sensitive alpha-amylase may be a hybrid of two or more calcium-sensitive alpha-amylases, such as a hybrid between the alpha-amylase of Bacillus amyloliquefaciens and the alpha-amylase of Bacillus licheniformis.

Commercially-available calcium-sensitive alpha-amylases are the products sold under

the following tradenames: Optitherm™ and Takatherm™ (available from Danisco); Maxamyl™ (available from Danisco), Spezym AA™, Spezyme Delta AA™, Spezyme Fred and Spezyme Xtra (available from Danisco), and Keistase™ (available from Daiwa), PURASTAR™ ST 5000E, and PURASTAR™ HPAM L (from Genencor Int.).

The A-, B-, C-, and carbohydrate binding domains of these calcium-sensitive alpha-amylases are provided in the following table:

C-terminal A-Domain extension or (A1 and A2 Carbohydrate Alpha-Amylase Domains) B-Domain C-Domain Binding Module Bacillus amyloliquefaciens 1-101 + 208-396 102-207 397-483 (SEQ ID NO: 1) Bacillus flavothermus 1-105 + 212-398 106-211 399-484 485-586 (SEQ ID NO: 2) Bacillus licheniformis 1-103 + 208-396 104-207 397-483 (SEQ ID NO: 3) Bacillus stearothermophilus 1-104 + 211-396 105-210 397-483 484-515 (SEQ ID NO: 4) Bacillus sp. (SEQ ID NO: 5) 1-105 + 213-398 106-212 399-485 Bacillus sp. NCIB 12512 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 6) Bacillus sp. NCIB 12513 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 7) SP707 (SEQ ID NO: 8) 1-105 + 213-398 106-212 399-485 TS-22 (SEQ ID NO: 9) 1-105 + 213-398 106-212 399-484 485-586 TS-23 (SEQ ID NO: 10) 1-105 + 213-398 106-212 399-484 485-583 Bacillus sp. KSM-AP1378 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 11) Bacillus sp. SP7-7 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 12) Bacillus stearothermophilus 1-104 + 211-396 105-210 397-483 484-486 alpha-amylase (Spezyme Xtra, SEQ ID NO: 29) Cytophaga alpha-amylase 1-102 + 209-397 103-208 398-484 (SEQ ID NO: 30)

Calcium-Insensitive Alpha-Amylases

Examples of calcium-insensitive alpha-amylases include the following:

1. Bacillus circulans alpha-amylase having the sequence of SEQ ID NO: 13;
2. KSM K-36 alpha-amylase having the sequence of SEQ ID NO: 14;
3. KSM K-38 alpha-amylase having the sequence of SEQ ID NO: 15;
4. Pyrococcus woesei alpha-amylase having the sequence of SEQ ID NO: 16;
5. Pyrococcus hybrid alpha-amylase described in WO 03/083054 having the amino acid sequence of SEQ ID NO: 31;
as well as hybrids and variants of any of these alpha-amylases.

The A-, B-, C-, and carbohydrate binding domains of these calcium-insensitive alpha-amylases are provided in the following table:

C-terminal extension or A-Domain Carbohydrate (A1 and A2 B- C- Binding Alpha-Amylase Domains) Domain Domain Module Bacillus circulans 1-102 + 209-395 103-208 396-482 483-492 (SEQ ID NO: 13) KSM K-36 1-103 + 208-393 104-207 394-480 (SEQ ID NO: 14) KSM K-38 1-103 + 208-393 104-207 394-480 (SEQ ID NO: 15) Pyrococcus 1-109 + 172-338 110-171 339-435 woesei (SEQ ID NO: 16) Pyrococcus 1-109 + 172-338 110-171 339-435 hybrid alpha-amylase (SEQ ID NO: 31)

The alpha-amylases may be produced by substituting the B-domain or a portion thereof of a calcium-sensitive alpha-amylase with the B-domain or a portion thereof of a calcium-insensitive alpha-amylase. The alpha-amylases also may be produced by substituting the A- and C-domains or a portion thereof of a calcium-insensitive alpha-amylase with the A- and C-domains or a portion thereof of a calcium-sensitive alpha-amylase. When producing a hybrid alpha-amylase, no amino acids should be deleted or inserted in the two splicing sites, i.e., the two sites where the sequence of the calcium-sensitive alpha-amylase is combined with the sequence of the calcium-insensitive alpha-amylase.

The boundaries of the A-, B- and C-domains of calcium-sensitive and calcium-insensitive amylases provided in the tables above are flexible, and some liberty regarding the sequences is permitted. Thus, in general it is possible to deviate from the exact boundaries for the domains by up to 20 amino acids, e.g., less than 20 amino acids, less than 10 amino acids, less than 6 amino acids, and less than 3 amino acids. In other words, the sequence of the calcium-sensitive alpha-amylase to be replaced with the sequence of a calcium-insensitive alpha-amylase may be within 20 amino acids of the boundaries of the B-domain, e.g., less than 10 amino acids, within 6 amino acids, and within 3 amino acids. For example, the boundaries differ by one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, or ten amino acids.

For example, for the B. amyloliquefaciens alpha-amylase (SEQ ID NO: 1) where the B-domain has been determined as amino acid residues 102-207, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 92-112 and ending at a position in the range of positions 197-217, e.g., starting at a position in the range of positions 96-108 and ending at a position in the range of positions 198-213 or starting at a position in the range of positions 99-105 and ending at a position in the range of positions 204-210. The A and C-domains of the B. amyloliquefaciens alpha-amylase were determined to be amino acid residues 1-101 (A1)+208-396 (A2) and 397-483, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 91-111, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 96-101 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 101-106. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 198-218 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 480-483.

For the B. flavothermus alpha-amylase (SEQ ID NO: 2) where the B-domain has been determined as amino acid residues 106-211, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 198-218, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 202-214 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 205-212. The A and C-domains of the B. flavothermus alpha-amylase were determined to be amino acid residues 1-105 (A1)+212-398 (A2) and 399-484, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 202-222 and ending at a position in the range of positions 479-484, e.g., starting at a position in the range of positions 207-212 and ending at a position in the range of positions 481-484 or starting at a position in the range of positions 212-217 and ending at a position in the range of positions 481-484. The B. flavothermus alpha-amylase further has a carbohydrate binding domain of amino acid residues 485-586. The carbohydrate binding domain is not required for the amylase activity and might be fully or partially deleted.

For the B. licheniformis alpha-amylase (SEQ ID NO: 3) where the B-domain has been determined as amino acid residues 104-207, the sequence of B. licheniformis alpha-amylase to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 94-114 and ending at a position in the range of positions 194-214, e.g., starting at a position in the range of positions 98-110 and ending at a position in the range of positions 198-210 or starting at a position in the range of positions 101-107 and ending at a position in the range of positions 201-207. The A and C-domains of the B. licheniformis alpha-amylase were determined to be amino acid residues 1-103 (A1)+208-396 (A2) and 397-483, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 93-113, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 98-103 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 103-108. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 198-218 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 480-483.

For the B. stearothermophilus alpha-amylase (SEQ ID NO: 4) where the B-domain has been determined as amino acid residues 105-210, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 95-115 and ending at a position in the range of positions 197-213, e.g., starting at a position in the range of positions 99-111 and ending at a position in the range of positions 201-213 or starting at a position in the range of positions 102-108 and ending at a position in the range of positions 204-210. The A and C-domains of the B. stearothermophilus alpha-amylase were determined to be amino acid residues 1-104 (A1)+211-396 (A2) and 397-483, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 94-114, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 99-104 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 201-221 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 206-211 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 211-216 and ending at a position in the range of positions 480-483. The B. stearothermophilus alpha-amylase further has a C-terminal extension of amino acid residues 484-586. The C-terminal extension is not required for the amylase activity and might be fully or partially deleted.

For the Bacillus alpha-amylase (SEQ ID NO: 5) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the Bacillus alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-396 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the Bacillus alpha-amylase (SEQ ID NO: 6) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the Bacillus alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the Bacillus alpha-amylase (SEQ ID NO: 7) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the Bacillus alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the SP707 alpha-amylase (SEQ ID NO: 8) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the SP707 alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the TS-22 alpha-amylase (SEQ ID NO: 9) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the TS-22 alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-484, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 481-484, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-484 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-484. The TS-22 alpha-amylase further has a carbohydrate binding domain of amino acid residues 485-586. The carbohydrate binding domain is not required for the amylase activity and might be fully or partially deleted.

For the TS-23 alpha-amylase (SEQ ID NO: 10) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the TS-23 alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-484, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-484, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-484 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-484. The TS-23 alpha-amylase further has a carbohydrate binding domain of amino acid residues 485-583. The carbohydrate binding domain is not required for the amylase activity and might be fully or partially deleted.

For the KSM-AP1378 alpha-amylase (SEQ ID NO: 11) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the KSM-AP1378 alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the Bacillus SP7-7 alpha-amylase (SEQ ID NO: 12) where the B-domain has been determined as amino acid residues 106-212, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 96-116 and ending at a position in the range of positions 199-219, e.g., starting at a position in the range of positions 100-112 and ending at a position in the range of positions 203-215 or starting at a position in the range of positions 103-109 and ending at a position in the range of positions 206-212. The A and C-domains of the Bacillus SP7-7 alpha-amylase were determined to be amino acid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485.

For the B. stearothermophilus alpha-amylase (SEQ ID NO: 29) where the B-domain has been determined as amino acid residues 105-210, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 95-115 and ending at a position in the range of positions 197-213, e.g., starting at a position in the range of positions 99-111 and ending at a position in the range of positions 201-213 or starting at a position in the range of positions 102-108 and ending at a position in the range of positions 204-210. The A and C-domains of the B. stearothermophilus alpha-amylase were determined to be amino acid residues 1-104 (A1)+211-396 (A2) and 397-483, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 94-114, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 99-104 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 201-221 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 206-211 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 211-216 and ending at a position in the range of positions 480-483. The B. stearothermophilus alpha-amylase further has a C-terminal extension of amino acid residues 484-486. The C-terminal extension is not required for the amylase activity and might be fully or partially deleted.

For the Cytophagus alpha-amylase (SEQ ID NO: 30) where the B-domain has been determined as amino acid residues 103-208, the sequence to be replaced by the corresponding sequence of a calcium-insensitive alpha-amylase starts at a position in the range of positions 93-113 and ending at a position in the range of positions 195-215, e.g., starting at a position in the range of positions 97-109 and ending at a position in the range of positions 199-211 or starting at a position in the range of positions 100-106 and ending at a position in the range of positions 202-208. The A and C-domains of the Cytophagus alpha-amylase were determined to be amino acid residues 1-102 (A1)+209-397 (A2) and 398-484, respectively. The alpha-amylases of the present invention may comprise an A1-domain starting at a position in the range of positions 1-5 and ending a position in the range of positions 92-112, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 97-102 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 102-107. The alpha-amylases of the present invention may comprise A2 and C-domains starting at a position in the range of positions 199-219 and ending at a position in the range of positions 479-484, e.g., starting at a position in the range of positions 204-209 and ending at a position in the range of positions 481-484 or starting at a position in the range of positions 209-214 and ending at a position in the range of positions 481-484.

For the Bacillus circulans alpha-amylase (SEQ ID NO: 13), the B-domain has been determined as amino acid residues 103-208. The alpha-amylases of the present invention may comprise a B-domain starting at a position in the range of positions 93-113 and ending at a position in the range of positions 195-215, e.g., starting at a position in the range of positions 97-109 and ending at a position in the range of positions 199-211 or starting at a position in the range of positions 100-106 and ending at a position in the range of positions 202-208. The A and C-domains of the Bacillus circulans alpha-amylase were determined to be amino acid residues 1-102 (A1)+209-395 (A2) and 396-482, respectively. The A1-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 1-5 and ending at a position in the range of positions 92-112, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 97-102 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 102-107. The A2-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 199-219 and ending at a position in the range of positions 385-405, e.g., starting at a position in the range of positions 204-209 and ending at a position in the range of positions 390-395 or starting at a position in the range of positions 209-214 and ending at a position in the range of positions 395-400. The A1 and A2 domains are preferably replaced simultaneously by the corresponding sequence of a calcium-sensitive alpha-amylase. The C-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 386-406 and ending at a position in the range of positions 477-482, e.g., starting at a position in the range of positions 391-396 and ending at a position in the range of positions 479-482 or starting at a position in the range of positions 396-401 and ending at a position in the range of positions 479-482. The Bacillus circulans alpha-amylase further has a C-terminal extension of amino acid residues 483-492. The extension is not required for the amylase activity and might be fully or partially deleted.

For the KSM-K36 alpha-amylase (SEQ ID NO: 14), the B-domain has been determined as amino acid residues 104-207. The alpha-amylases of the present invention may comprise a B-domain starting at a position in the range of positions 93-113 and ending at a position in the range of positions 195-215, e.g., starting at a position in the range of positions 97-109 and ending at a position in the range of positions 199-211 or starting at a position in the range of positions 100-106 and ending at a position in the range of positions 202-208. The A and C-domains of the Bacillus circulans alpha-amylase were determined to be amino acid residues 1-103 (A1)+208-393 (A2) and 394-480, respectively. The A1-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 1-5 and ending at a position in the range of positions 93-113, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 98-103 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 103-108. The A2-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 198-218 and ending at a position in the range of positions 383-403, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 388-393 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 393-398. The A1 and A2 domains are preferably replaced simultaneously by the corresponding sequence of a calcium-sensitive alpha-amylase. The C-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 384-404 and ending at a position in the range of positions 475-480, e.g., starting at a position in the range of positions 389-394 and ending at a position in the range of positions 477-480 or starting at a position in the range of positions 394-399 and ending at a position in the range of positions 477-480.

For the KSM-K38 alpha-amylase (SEQ ID NO: 15), the B-domain has been determined as amino acid residues 104-207. The alpha-amylases of the present invention may comprise a B-domain starting at a position in the range of positions 93-113 and ending at a position in the range of positions 195-215, e.g., starting at a position in the range of positions 97-109 and ending at a position in the range of positions 199-211 or starting at a position in the range of positions 100-106 and ending at a position in the range of positions 202-208. The A and C-domains of the Bacillus circulans alpha-amylase were determined to be amino acid residues 1-103 (A1)+208-393 (A2) and 394-480, respectively. The A1-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 1-5 and ending at a position in the range of positions 93-113, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 98-103 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 103-108. The A2-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 198-218 and ending at a position in the range of positions 383-403, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 388-393 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 393-398. The A1 and A2 domains are preferably replaced simultaneously by the corresponding sequence of a calcium-sensitive alpha-amylase. The C-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 384-404 and ending at a position in the range of positions 475-480, e.g., starting at a position in the range of positions 389-394 and ending at a position in the range of positions 477-480 or starting at a position in the range of positions 394-399 and ending at a position in the range of positions 477-480.

For the Pyrococcus woesei alpha-amylase (SEQ ID NO: 16), the B-domain has been determined as amino acid residues 110-171. The alpha-amylases of the present invention may comprise a B-domain starting at a position in the range of positions 100-120 and ending at a position in the range of positions 161-181, e.g., starting at a position in the range of positions 105-115 and ending at a position in the range of positions 166-171 or starting at a position in the range of positions 107-113 and ending at a position in the range of positions 171-176. The A and C-domains of the Bacillus circulans alpha-amylase were determined to be amino acid residues 1-109 (A1)+172-338 (A2) and 339-435, respectively. The A1-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 1-5 and ending at a position in the range of positions 99-119, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 109-114. The A2-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 161-181 and ending at a position in the range of positions 328-348, e.g., starting at a position in the range of positions 167-172 and ending at a position in the range of positions 333-338 or starting at a position in the range of positions 172-177 and ending at a position in the range of positions 338-343. The A1 and A2 domains are preferably replaced simultaneously by the corresponding sequence of a calcium-sensitive alpha-amylase. The C-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 329-349 and ending at a position in the range of positions 430-435, e.g., starting at a position in the range of positions 324-329 and ending at a position in the range of positions 432-435 or starting at a position in the range of positions 329-344 and ending at a position in the range of positions 432-435.

For the Pyrococcus hybrid alpha-amylase (SEQ ID NO: 31), the B-domain has been determined as amino acid residues 110-171. The alpha-amylases of the present invention may comprise a B-domain starting at a position in the range of positions 100-120 and ending at a position in the range of positions 161-181, e.g., starting at a position in the range of positions 105-115 and ending at a position in the range of positions 166-171 or starting at a position in the range of positions 107-113 and ending at a position in the range of positions 171-176. The A and C-domains of the Bacillus circulans alpha-amylase were determined to be amino acid residues 1-109 (A1)+172-338 (A2) and 339-435, respectively. The A1-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 1-5 and ending at a position in the range of positions 99-119, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 109-114. The A2-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 161-181 and ending at a position in the range of positions 328-348, e.g., starting at a position in the range of positions 167-172 and ending at a position in the range of positions 333-338 or starting at a position in the range of positions 172-177 and ending at a position in the range of positions 338-343. The A1 and A2 domains are preferably replaced simultaneously by the corresponding sequence of a calcium-sensitive alpha-amylase. The C-domain which can be replaced by the corresponding sequence of a calcium-sensitive alpha-amylase starts at a position in the range of positions 329-349 and ending at a position in the range of positions 430-435, e.g., starting at a position in the range of positions 334-339 and ending at a position in the range of positions 432-435 or starting at a position in the range of positions 339-344 and ending at a position in the range of positions 432-435.

Variants

The variants of the present invention have alpha-amylase activity and comprise A-, B-, and C-domains and, optionally, a carbohydrate-binding module.

A-Domain of the Variants

The variants of the present invention comprise an A-domain of a calcium-sensitive alpha-amylase.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 1.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 2.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 3.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 4.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 5.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 6.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 7.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 8.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 9.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 10.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 11.

In an embodiment, the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 12.

In an embodiment, the A-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 29.

In an embodiment, the A-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 30.

B-Domain of the Variants

The variants of the present invention comprise a B-domain which has at least 55% and less than 100% sequence identity with the B-domain of SEQ ID NO: 13. For example, the B-domain may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the B-domain of SEQ ID NO: 13.

The amino acid sequence of the B-domain has at least one difference, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at a position corresponding to position 105, 115, 117, 129, 132, 134, 135, 150, 157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177, 179, 180, 181, 182, 184, 187, 188, 191, 202, 204, 206, 208, or 210.

In an embodiment, the B-domain has 1-15 differences compared with the B-domain of SEQ ID NO: 13, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 differences.

In an embodiment, the amino acid sequence of the B-domain has at least one difference, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at a position corresponding to position 105, 115, 117, 129, 132, 134, 135, 150, 157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177, 184, 187, 188, 191, 202, 204, 206, 208, or 210, and the amino acids at at least two positions corresponding to positions 179-182 are absent.

In an embodiment, a difference may be at the position corresponding to position 105, i.e., the amino acid is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.

In an embodiment, a difference may be at the position corresponding to position 115, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, e.g., Trp.

In an embodiment, a difference may be at the position corresponding to position 117, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.

In an embodiment, a difference may be at the position corresponding to position 129, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Val.

In an embodiment, a difference may be at the position corresponding to position 132, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.

In an embodiment, a difference may be at the position corresponding to position 134, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu or Tyr.

In an embodiment, a difference may be at the position corresponding to position 135, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn or Gln.

In an embodiment, a difference may be at the position corresponding to position 150, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr.

In an embodiment, a difference may be at the position corresponding to position 157, i.e., the amino acid is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.

In an embodiment, a difference may be at the position corresponding to position 159, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.

In an embodiment, a difference may be at the position corresponding to position 160, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.

In an embodiment, a difference may be at the position corresponding to position 164, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, e.g., Val.

In an embodiment, a difference may be at the position corresponding to position 166, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp.

In an embodiment, a difference may be at the position corresponding to position 168, i.e., the amino acid is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu.

In an embodiment, a difference may be at the position corresponding to position 169, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ser.

In an embodiment, a difference may be at the position corresponding to position 170, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg.

In an embodiment, a difference may be at the position corresponding to position 171, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Lys.

In an embodiment, a difference may be at the position corresponding to position 172, i.e., the amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.

In an embodiment, a difference may be at the position corresponding to position 174, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg.

In an embodiment, a difference may be at the position corresponding to position 176, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.

In an embodiment, a difference may be at the position corresponding to position 177, i.e., the amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.

In an embodiment, a difference may be at the position corresponding to position 179, i.e., the amino acid is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn, Asp, Gln, or Glu, or is absent.

In an embodiment, a difference may be at the position corresponding to position 180, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.

In an embodiment, a difference may be at the position corresponding to position 181, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Asp, Cys, Leu, or Pro, or is absent.

In an embodiment, a difference may be at the position corresponding to position 182, i.e., the amino acid is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.

In an embodiment, a difference may be at the position corresponding to position 184, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala.

In an embodiment, a difference may be at the position corresponding to position 187, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp.

In an embodiment, a difference may be at the position corresponding to position 188, i.e., the amino acid is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu.

In an embodiment, a difference may be at the position corresponding to position 191, i.e., the amino acid is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr.

In an embodiment, a difference may be at the position corresponding to position 202, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.

In an embodiment, a difference may be at the position corresponding to position 204, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, e.g., Lys or Met.

In an embodiment, a difference may be at the position corresponding to position 206, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, e.g., Lys or Met.

In an embodiment, a difference may be at the position corresponding to position 208, i.e., the amino acid is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.

In an embodiment, a difference may be at the position corresponding to position 210, i.e., the amino acid is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr or Val.

In an embodiment, the amino acids at the positions corresponding to positions 179 and 180 are absent.

In an embodiment, the amino acids at the positions corresponding to positions 179 and 181 are absent.

In an embodiment, the amino acids at the positions corresponding to positions 179 and 182 are absent.

In an embodiment, the amino acids at the positions corresponding to positions 180 and 181 are absent.

In an embodiment, the amino acids at the positions corresponding to positions 180 and 182 are absent.

In an embodiment, the amino acids at the positions corresponding to positions 181 and 182 are absent.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 117, 150, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 129, 177, and 179.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 132, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 134, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 135, 179, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105 and 150.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, 164, 166, 168, 171, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, 164, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, 166, 168, and 171.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, 166, 168, 171, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 150, 184, and 206.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 157, 159, 160, 184, 208, and 210.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 160, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 164, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 166, 168, and 171.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 179, and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105 and 184.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105, 184, and 210.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 105 and 206.

In an embodiment, the amino acid sequence of the B-domain of the variant is different from the amino acid sequence of SEQ ID NO: 13 at the positions corresponding to positions 187, 188, and 191.

The alpha-amylases of the invention may further comprise additional substitutions, insertions or deletions in the B-domain derived from the calcium-insensitive alpha-amylase. Examples of suitable substitutions, insertions or deletions in the B-domain of a calcium-insensitive alpha-amylase are the alterations corresponding to the following alterations in B. circulans alpha-amylase: E179*, N180*, E185W, N186E and D189T (SEQ ID NO: 13 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering.

In another embodiment, the alpha-amylases of the present invention comprise the substitution Q150T.

In another embodiment, the alpha-amylases of the present invention comprise the substitution T164V.

In another embodiment, the alpha-amylases of the present invention comprise the substitution K184A.

C-Domain of the Variants

The variants of the present invention comprise a C-domain of a calcium-sensitive alpha-amylase.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 1.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 2.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 3.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 4.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 5.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 6.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 7.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 8.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 9.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 10.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 11.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 12.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 29.

In an embodiment, the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 30.

In an embodiment, the alpha-amylase variants have a ratio of activity measured by the Phadebas activity to the activity measured by the G7-pNG assay greater than 0.1, preferably of more than 0.25, even more preferred more than 0.5 and most preferred more than 1.

The Phadebas assay is an assay for determining alpha-amylase activity using a cross-linked insoluble blue-colored starch polymer. (Phadebas® Amylase Test, supplied by Magle Life Sciences, Lund, Sweden).

The G7-pNG assay is an assay for determining alpha-amylase activity using a soluble chromogen compound, p-nitrophenyl-alpha-D-maltoheptaoside. Kits containing PNP-G7 substrate and alpha-glucosidase is manufactured by Boehringer-Mannheim (cat. no. 1054635).

In order to determine the amylase activity using the Phadebas and the G7-pNG assays, a reference amylase with known activity must be included in the assay and the activity is determined relative to the known reference. For purposes of the present invention, the reference alpha-amylase which is considered to have the same activity when measured by the Phadebas and the G7-pNG assays, is the Bacillus licheniformis alpha-amylase sold by Novozymes A/S under the tradename Termamyl®, which has the sequence of SEQ ID NO: 3. Thus, the reference alpha-amylase has a ratio of 1 when measuring the activity by the Phadebas assay relative to the activity measured by the G7-pNP assay.

The ratio of activity on insoluble substrate to activity on soluble substrate is determined by measuring the activities on the two particular selected substrates and calculating the ratio. Preferably the ratio is at least 1.5 fold higher than for the calcium-insensitive alpha-amylase, e.g., at least 2 fold higher, at least 2.5 fold higher and at least 3 fold higher.

Using the methods disclosed below for determining the ratio of activity by the Phadebas assay to the activity of the G7pNG assay, the B. circulans alpha-amylase having the amino acid sequence of SEQ ID NO: 13 was found to have a ratio of approximately 0.014.

Additional Mutation(s)

The alpha-amylases of the invention may comprise one or more substitutions, insertions and/or deletions known in the art to improve the properties of alpha-amylases.

For example, oxidizable amino acid residues may be substituted with a non-oxidizable amino acid residue in order to improve the stability of the enzyme under oxidizing conditions, e.g., in the presence of bleach, in accordance with the teachings of WO 94/02597 and WO 94/18314, which are incorporated herein by reference.

Further beneficial substitutions that may be introduced are disclosed in WO 99/23211, WO 01/66712 and WO 2006/002643, which are incorporated herein by reference.

The variants of the present invention preferably consist of 481 to 515, 481 to 493, or 481 to 486 amino acids.

Preparation of Variants

The present invention also relates to methods for obtaining a variant having alpha-amylase activity, comprising: (a) introducing into a parent alpha-amylase a substitution at one or more (several) positions corresponding to positions 105, 115, 117, 129, 132, 134, 135, 150, 157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177, 179, 180, 181, 184, 187, 188, 191, 206, 208, and 210, wherein the variant has alpha-amylase activity; and (b) recovering the variant.

The variants can be prepared according to any mutagenesis procedure known in the art, such as site-directed mtagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several) mutations are created at a defined site in a polynucleotide molecule encoding the parent alpha-amylase. The technique can be performed in vitro or in vivo.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide molecule of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein olgionucleotides are synthesized and assembled upon photo-programable microfluidic chips.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent alpha-amylase and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Research 18: 7349-4966.

Site-directed mutagenesis can be accomplished in vivo by methods known in the art.

See, for example, U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnology 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants of a parent alpha-amylase.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic constuction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplfied using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR ampflication. Polynucleotide fragments may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides that encode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

An isolated polynucleotide encoding a variant may be manipulated in a variety of ways to provide for expression of the variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized by a host cell for expression of the polynucleotide. The promoter sequence contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter including a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include modified promoters including the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used in the present invention.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the polypeptide-encoding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a variant and directs the encoded polypeptide into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the variant. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell may be used in the present invention.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences described above may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase production of an alpha-amylase variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra) to obtain substantially pure alpha-amylase variants.

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant, which are advantageously used in the recombinant production of the variant. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive bacterium or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

In one aspect, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

In another aspect, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In another aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In another aspect, the yeast host cell is a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

In another aspect, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In another aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In another aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant from the cultivation medium.

The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing a variant is used as a source of the variant.

The variant may be detected using methods known in the art that are specific for the variants. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein in the Examples.

The variant may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

A variant of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

Plants

The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising an isolated polynucleotide encoding a variant of the present invention so as to express and produce the variant in recoverable quantities. The variant may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant variant may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seed coats.

Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant of the present invention may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (several) expression constructs encoding the variant into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding the variant operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the variant is desired to be expressed. For instance, the expression of the gene encoding a variant may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higher expression of a polypeptide of the present invention in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428. Additional transformation methods for use in accordance with the present disclosure include those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by reference in their entirety).

Following transformation, the transformants comprising the expression construct are selected and regenerated into whole plants according to methods well known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype with a construct prepared according to the present invention, transgenic plants may be made by crossing a plant having a construct of the present invention to a second plant lacking the construct. For example, a construct encoding a variant or a portion thereof can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present invention not only encompasses a plant directly regenerated from cells which have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a DNA construct prepared in accordance with the present invention, or a portion of a DNA construct prepared in accordance with the present invention. Crossing results in a transgene of the present invention being introduced into a plant line by cross pollinating a starting line with a donor plant line that includes a transgene of the present invention. Non-limiting examples of such steps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

The present invention also relates to methods of producing a variant of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and (b) recovering the variant.

Compositions

The present invention also relates to compositions comprising an alpha-amylase variant and at least one additional enzyme. The additional enzyme(s) may be selected from the group consisting of beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase), glucoamylase, hemicellulsae (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, pullulanase, and/or other enzymes useful in a commercial process in conjunction with an alpha-amylase. The additional enzyme may also be a second alpha-amylase. Such enzymes are known in the art in starch processing, sugar conversion, fermentations for alcohol and other useful end-products, commercial detergents and cleaning aids, stain removal, fabric treatment or desizing, and the like.

Methods of Using the Alpha-Amylase Variants—Industrial Applications

The variants of the present invention possess valuable properties allowing for a variety of industrial applications. In particular, the variants may be used in detergents, in particular laundry detergent compositions and dishwashing detergent compositions, hard surface cleaning compositions, and for desizing textiles, fabrics or garments, production of pulp and paper, beer making, ethanol production, and starch conversion processes.

The alpha-amylase variants may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, and WO 96/28567, which are all hereby incorporated by reference). Also contemplated are compositions for starch conversion purposes, which may beside the variant of the invention also comprise an AMG, pullulanase, and other alpha-amylases.

Further, the variants are particularly useful in the production of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which is hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains.

The variants may also be used for desizing of textiles, fabrics, and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP 119920, which are incorporated herein by reference), beer making or brewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this “gelatinization” process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or “liquefied” so that it can be suitably processed. This reduction in viscosity is primarily attained by enzymatic degradation in current commercial practice.

Conventional starch-conversion processes, such as liquefaction and saccharification processes are described, e.g., in U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, which are incorporated herein by reference.

In an embodiment, the conversion process degrading starch to lower molecular weight carbohydrate components such as sugars or fat replacers includes a debranching step.

In the case of converting starch into a sugar, the starch is depolymerized. Such a depolymerization process consists of, e.g., a pre-treatment step and two or three consecutive process steps, i.e., a liquefaction process, a saccharification process, and depending on the desired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can be separated into 4 main steps: milling, liquefaction, saccharification, and fermentation.

The grain is milled in order to open up the structure and allow for further processing. Two processes used are wet or dry milling. In dry milling, the whole kernel is milled and used in the remaining part of the process. Wet milling gives a very good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized by hydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysis may be carried out by acid treatment or enzymatically by an alpha-amylase. Acid hydrolysis is used on a limited basis. The raw material can be milled whole grain or a side stream from starch processing.

During a typical enzymatic liquefaction, the long-chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase. Enzymatic liquefaction is generally carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C. (e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are) added. The liquefaction process is carried out at 85° C. for 1-2 hours. The pH is generally between 5.5 and 6.2. In order to ensure optimal enzyme stability under these conditions, 1 mM of calcium is added (to provide about 40 ppm free calcium ions). After such treatment, the liquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g., 105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added to obtain the final hydrolysis. The liquefaction process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefied grain is also known as mash.

Liquefied starch-containing material is saccharified in the presence of saccharifying enzymes such as glucoamylases. The saccharification process may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to 60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to 65° C., typically around 60° C., which is followed by a complete saccharification during fermentation referred to as simultaneous saccharification and fermentation (SSF). The pH is usually between 4.2-4.8, e.g., 4.5. In a simultaneous saccharification and fermentation (SSF) process, there is no holding stage for saccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced during liquefaction are converted into dextrose by adding a glucoamylase and a debranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase. The temperature is lowered to 60° C., prior to the addition of a glucoamylase and debranching enzyme. The saccharification process proceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced to below 4.5, while maintaining a high temperature (above 95° C.), to inactivate the liquefying alpha-amylase. This process reduces the formation of short oligosaccharide called “panose precursors,” which cannot be hydrolyzed properly by the debranching enzyme. Normally, about 0.2-0.5% of the saccharification product is the branched trisaccharide panose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by a pullulanase. If active amylase from the liquefaction remains present during saccharification (i.e., no denaturing), the amount of panose can be as high as 1-2%, which is highly undesirable since it lowers the saccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularly glucose) are produced from enzymatic saccharification. These fermentable sugars may be further purified and/or converted to useful sugar products. In addition, the sugars may be used as a fermentation feedstock in a microbial fermentation process for producing end-products, such as alcohol (e.g., ethanol and butanol), organic acids (e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during the liquefaction process steps are used to produce alcohol and particularly ethanol. In ethanol production, an SSF process is commonly used wherein the saccharifying enzymes and fermenting organisms (e.g., yeast) are added together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desired end-product. Typically, if ethanol is the desired end product yeast will be used as the fermenting organism. In some preferred embodiments, the ethanol-producing microorganism is a yeast and specifically Saccharomyces such as strains of S. cerevisiae (U.S. Pat. No. 4,316,956). A variety of S. cerevisiae are commercially available and these include but are not limited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China). The amount of starter yeast employed in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g., to produce at least 10% ethanol from a substrate having between 25-40% DS in less than 72 hours). Yeast cells are generally supplied in amounts of about 104 to about 1012, and preferably from about 107 to about 1010 viable yeast count per mL of fermentation broth. After yeast is added to the mash, it is typically subjected to fermentation for about 24-96 hours, e.g., 35-60 hours. The temperature is between about 26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g., around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms (e.g., yeast), nutrients, and additional enzymes, including phytases. The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, as is known in the art, can result in fermentation end product including, e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids, and derivatives thereof. More specifically when lactic acid is the desired end product, a Lactobacillus sp. (L. casei) may be used; when glycerol or 1,3-propanediol are the desired end-products E. coli may be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, Pantoea citrea may be used as the fermenting microorganism. The above enumerated list are only examples and one skilled in the art will be aware of a number of fermenting microorganisms that may be used to obtain a desired end product.

Processes for Producing Fermentation Products from Ungelatinized Starch-Containing Material

The invention relates to processes for producing fermentation products from starch-containing material without gelatinization (i.e., without cooking) of the starch-containing material. The fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water. In one embodiment a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s) to produce sugars that can be fermented into the fermentation product by a suitable fermenting organism. In this embodiment the desired fermentation product, e.g., ethanol, is produced from ungelatinized (i.e., uncooked), preferably milled, cereal grains, such as corn. Accordingly, in the first aspect the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material. In an embodiment a protease is also present. The protease may be any acid fungal protease or metalloprotease. The fermentation product, e.g., ethanol, may optionally be recovered after fermentation, e.g., by distillation. Typically amylase(s), such as glucoamylase(s) and/or other carbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are) present during fermentation. Examples of glucoamylases and other carbohydrate-source generating enzymes include raw starch hydrolyzing glucoamylases. Examples of alpha-amylase(s) include acid alpha-amylases such as acid fungal alpha-amylases. Examples of fermenting organisms include yeast, e.g., a strain of Saccharomyces cerevisiae. The term “initial gelatinization temperature” means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Stärke 44(12): 461-466. Before initiating the process a slurry of starch-containing material, such as granular starch, having 10-55, e.g., 25-45 and 30-40, w/w % dry solids (DS) of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol. %, preferably 15-60 vol. %, especially from about 30 to 50 vol. % water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like. The starch-containing material may be prepared by reducing the particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolyzate. A process in this aspect of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature typically lies in the range between 30-75° C., preferably between 45-60° C. In a preferred embodiment the process carried at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around 32° C. In an embodiment the process is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %, such as below about 1 w/w %, such as below about 0.5 w/w %, or below 0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 w/w %, such as below about 0.2 w/w %. The process of the invention may be carried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from Gelatinized Starch-Containing Material

In this aspect the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to processes for producing fermentation products from starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha-amylase variant, or;

(b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is also used in the liquefaction step. In an embodiment a protease, such as an acid fungal protease or a metallo protease is added before, during and/or after liquefaction. In an embodiment the metalloprotease is derived from a strain of Thermoascus, e.g., a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In an embodiment the carbohydrate-source generating enzyme is a glucoamylase derived from a strain of Aspergillus, e.g., Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, e.g., Trametes cingulata; a strain of the genus Pachykytospora, e.g., a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, e.g., Leucopaxillus giganteus; or a strain of the genus Peniophora, e.g., a strain of the species Peniophora rufomarginata; or a mixture thereof. Saccharification step (b) and fermentation step (c) may be carried out either sequentially or simultaneously. A pullulanase and/or metalloprotease may be added during saccharification and/or fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out simultaneously (SSF process). The pullulanase and/or metalloprotease may also advantageously be added before liquefaction (pre-liquefaction treatment), i.e., before or during step (a), and/or after liquefaction (post liquefaction treatment), i.e., after step (a). The pullulanase is most advantageously added before or during liquefaction, i.e., before or during step (a). The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. The fermenting organism is preferably yeast, preferably a strain of Saccharomyces cerevisiae. In a particular embodiment, the process of the invention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material, preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In an embodiment the particle size is smaller than a #7 screen, e.g., a #6 screen. A #7 screen is usually used in conventional prior art processes. The aqueous slurry may contain from 10-55 w/w % dry solids (DS), e.g., 25-45 and 30-40 w/w % dry solids (DS) of starch-containing material. The slurry is heated to above the gelatinization temperature and an alpha-amylase variant may be added to initiate liquefaction (thinning). The slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a). Liquefaction may in an embodiment be carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C., preferably between 70-90° C., such as preferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, and alpha-amylase variant, optionally together with a pullulanase and/or protease, preferably metalloprotease, are added to initiate liquefaction (thinning). In an embodiment the slurry may then be jet-cooked at a temperature between 95-140° C., preferably 100-135° C., such as 105-125° C., for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes. The slurry is cooled to 60-95° C. and more alpha-amylase variant and optionally pullulanase variant and/or protease, preferably metalloprotease, is(are) added to finalize hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5. Saccharification step (b) may be carried out using conditions well known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at temperatures from 20-75° C., preferably from 40-70° C., typically around 60° C., and at a pH between 4 and 5, normally at about pH 4.5. The most widely used process to produce a fermentation product, especially ethanol, is a simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that a fermenting organism, such as yeast, and enzyme(s), may be added together. SSF may typically be carried out at a temperature from 25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferably around about 32° C. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylase variants may also be used in a beer-making process and similar fermentations; the alpha-amylases will typically be added during the mashing process. The process is substantially similar to the milling, liquefaction, saccharification, and fermentation processes described above.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycled thin-stillage resulting in an aqueous slurry. The slurry can comprise between 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, 20 to 35% and 30-36% ds). In some embodiments, the recycled thin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to 50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water and backset, the pH is not adjusted in the slurry. Further the pH is not adjusted after the addition of a phytase and optionally an alpha-amylase to the slurry. In an embodiment, the pH of the slurry will be in the range of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and 5.2 depending on the amount of thin stillage added to the slurry and the type of material comprising the thin stillage. For example, the pH of the thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in the beer well, to reduce the risk of microbial contamination prior to distillation.

In some embodiments, a phytase is added to the slurry. In other embodiments, in addition to a phytase, an alpha-amylase is added to the slurry. In some embodiments, a phytase and alpha-amylase are added to the slurry sequentially. In other embodiments, a phytase and alpha-amylase are added simultaneously. In some embodiments, the slurry comprising a phytase and optionally, an alpha-amylase, are incubated (pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15 minutes to 4 hours). In other embodiments, the slurry is incubated at a temperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50 to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70 to 85° C. and 77 to 86° C.). In other embodiments, the slurry is incubated at a temperature of about 0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10° C. and 0 to 5° C.) below the starch gelatinization temperature of the starch-containing material. In some embodiments, the temperature is below about 68° C., below about 65° C., below about 62° C., below about 60° C. and below about 55° C. In some embodiments, the temperature is above about 45° C., above about 50° C., above about 55° C. and above about 60° C. In some embodiments, the incubation of the slurry comprising a phytase and an alpha-amylase at a temperature below the starch gelatinization temperature is referred to as a primary (1°) liquefaction.

In one embodiment, the milled starch-containing material is corn or milo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8 to 5.2, and the slurry is incubated with a phytase and optionally an alpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to 75° C.

Currently, it is believed that commercially-available microbial alpha-amylases used in the liquefaction process are generally not stable enough to produce liquefied starch substrate from a dry mill process using whole ground grain at a temperature above about 80° C. at a pH level that is less than pH 5.6. The stability of many commercially available alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreated starch-containing material is exposed to an increase in temperature such as about 0 to about 45° C. above the starch gelatinization temperature of the starch-containing material (e.g., 70° C. to 120° C., 70° C. to 110° C., and 70° C. to 90° C.) for a period of time of about 2 minutes to about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1 hour to 2 hours. The temperature can be increased by a conventional high temperature jet cooking system for a short period of time, for example, for 1 to 15 minutes. Then the starch maybe further hydrolyzed at a temperature ranging from about 75° C. to 95° C. (e.g., 80° C. to 90° C. and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30 to 120 minutes). In a preferred embodiment, the pH is not adjusted during these process steps and the pH of the liquefied mash is in the range of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, and pH 5.0 to 5.2). In some embodiments, a second dose of thermostable alpha-amylase is added to the secondary liquefaction step, but in other embodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed by saccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may be extracted by, for example, distillation and optionally followed by one or more process steps.

In some embodiments, the yield of ethanol produced by the methods provided herein is at least 8%, at least 10%, at least 12%, at least 14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) and at least 23% v/v. The ethanol obtained according to the process provided herein may be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically used for animal feed either in liquid or dried form. In further embodiments, the end product may include the fermentation co-products such as distiller's dried grains (DDG) and distiller's dried grain plus solubles (DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to the skilled person.

According to the process provided herein, the saccharification and fermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylase variants may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where re-pulping occurs at pH above 7 and where amylases facilitate the disintegration of the waste material through degradation of the reinforcing starch. The alpha-amylase variants are especially useful in a process for producing a papermaking pulp from starch-coated printed-paper. The process may be performed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after step a), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylase variants may also be useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alpha-amylase variants it is possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

The alpha-amylase variants may also be very useful in textile, fabric or garment desizing. In the textile processing industry, alpha-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size, which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the fiber material. In order to reduce processing cost and increase mill throughput, the desizing process is sometimes combined with the scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size leads to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the alpha-amylase variants as they have an improved performance in alkaline solutions. The alpha-amylase variants may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.

Desizing and bleaching processes are well known in the art. For instance, such processes are described in, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, EP 119920, which are hereby incorporated by reference.

Cleaning Processes and Detergent Compositions

The alpha-amylase variants may be added as a component of a detergent composition for various cleaning or washing processes, including laundry and dishwashing. For example, the variants may be used in the detergent compositions described in WO 96/23874 and WO 97/07202.

The alpha-amylase variants may be incorporated in detergents at conventionally employed concentrations. For example, a variant of the invention may be incorporated in an amount corresponding to 0.00001-10 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.

The detergent composition may for example be formulated as a hand or machine laundry detergent composition, including a laundry additive composition suitable for pretreatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more other enzymes, such as a lipase, peroxidase, protease, another amylolytic enzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase, CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes, Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatible with the selected detergent (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive, e.g., a separate additive or a combined additive, can be formulated, e.g., granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols, fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to about 70% water and 0 to about 30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from about 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleiclacrylic acid copolymers and lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system may comprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, e.g., WO 92/19708 and WO 92/19709.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.055 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

One or more of the variant enzymes described herein may additionally be incorporated in the detergent formulations disclosed in WO 97/07202, which is hereby incorporated as reference.

This disclosure includes further detail in the following examples, which are not in any way intended to limit the scope of what is claimed. The following examples are thus offered to illustrate, but not to limit what is claimed.

EXAMPLES Materials and methods Fermentation of Alpha-Amylases and Variants

Fermentation may be performed by methods well known in the art or as follows:

A B. subtilis strain harboring the relevant expression plasmid is streaked on a LB-agar plate with a relevant antibiotic, and grown overnight at 37° C.

The colonies are transferred to 100 ml BPX media supplemented with a relevant antibiotic (for instance 10 mg/l chloroamphinicol) in a 500 ml shaking flask. Composition of BPX medium:

Potato starch 100 g/l  Barley flour 50 g/l BAN 5000 SKB 0.1 g/l  Sodium caseinate 10 g/l Soy Bean Meal 20 g/l Na2HPO4, 12H2O  9 g/l Pluronic ™ 0.1 g/l 

BAN is a Bacillus amyloliquefaciens alpha-amylase product sold by Novozymes.

The culture is shaken at 37° C. at 270 rpm for 4 to 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatant is filtered to obtain a completely clear solution. The filtrate is concentrated and washed on an UF-filter (10000 cut off membrane) and the buffer is changed to 20 mM acetate pH 5.5. The UF-filtrate is applied on an S-sepharose F.F. and elution is carried out by step elution with 0.2 M NaCl in the same buffer. The eluate is dialyzed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3 M NaCl over 6 column volumes. The fractions, which contain the activity (measured by the Phadebas assay) are pooled, pH is adjusted to 7.5 and remaining color is removed by a treatment with 0.5% w/vol active coal in 5 minutes.

Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Magle Life Sciences, Lund, Sweden) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 mL 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl2, 0.01% TRITON® X100, pH adjusted to the value of interest with NaOH). This is the substrate solution. The alpha-amylase to be tested is diluted in 50 mM Britton-Robinson buffer. This is the amylase solution. The test is performed at constant temperature, e.g., at room temperature, 37° C. or 50° C. The insoluble blue-colored starch polymer is hydrolyzed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity.

575 microliters substrate solution is equilibrated at the selected temperature for 5 minutes. The hydrolysis is started by adding 25 microliters amylase solution to the substrate solution and incubating the sample under gentle mixing for 15 minutes at the selected temperature. The reaction is stopped by adding 100 microliters 1 M NaOH and immediately cooling on an ice bath after mixing. After centrifugation at 500 gav for 3 minutes, 200 microliters of the supernatant is transferred to a microtiter plate, and the absorbance at 620 nm is read (Aamyl). The blind is prepared as described but where the 25 microliters amylase solution is replaced by 25 microliters 50 mM Britton-Robison buffer. The absorbance of the blind at 620 nm is Ab. The standard curve is prepared similarly by making a dilution series of Termamyl with a known activity and measuring the release of blue color to the solution as described above. The absorbance of the standards at 620 nm is As. The standard curve is a plot of As-Ab against the Termamyl activity in the sample. The activity of the amylase of interest can be determined by comparing Aamyl-Ab to the Termamyl standard curve.

It is important that the measured 620 nm absorbance after 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme (both amylase of interest and standard) must therefore be adjusted to fit this criterion. Under a specified set of conditions (temperature, pH, reaction time, buffer conditions), 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue color will be produced.

G7-pNP Amylase Assay

Alpha-amylase activity may also be determined by a method employing the PNP-G7 substrate. PNP-G7 which is an abbreviation for p-nitrophenyl-alpha-D-maltoheptaoside, is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the alpha-glucosidase included in the kit digests the substrate to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophometry at 2=405 nm (400-420 nm). Kits containing PNP-G7 substrate and alpha-glucosidase is manufactured by Roche/Hitachi (cat. no.11876473). The G7-PNP substrate from this kit contains 22 mmol/L 4,6-ethylidene-G7-PNP and 52.4 mmol/L HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0) and the alpha-glucosidase contains 52.4 mmol/L HEPES, 87 mmol/L NaCl, 12.6 mmol/L MgCl2, 0.075 mmol/L CaCl2, ≧4 kU/L alpha-glucosidase).

The amylase sample to be analyzed is diluted in 50 mM EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Sigma, E9502)), 0.01% TRITON® X100, 1 mM CaCl2, pH 7.0. Before use substrate working solution was made by mixing 1 mL of the alpha-glucosidase containing reagent with 0.2 mL 4,6-ethylidene-G7-PNP containing reagent from kit. Immediately after incubation of samples in PCR machine the samples are diluted 10 times in residual activity buffer (50 mM EPPS, 0.01% TRITON® X100, 1 mM CaCl2, pH7.0). The assay is performed by transferring 20 microliters diluted enzyme samples to a 96 well microtiter plate and adding 80 microliters substrate working solution. The solution is mixed and pre-incubated 1 minute at room temperature and absorption is measured every 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions. The amylase sample should be diluted to a level where the slope is below 0.4 absorbance units per minute.

Enzchek® Amylase Activity Assay

Alpha-amylase activity may also be determined by a method employing the EnzChek® substrate. The substrate in the EnzChek® Ultra Amylase Assay Kit (E33651, Invitrogen, La Jolla, Calif., USA) is a corn starch derivative, DQ™ starch, which is corn starch labeled with BODIPY® FL dye to such a degree that fluorescence is quenched.

One vial containing approx. 1 mg lyophilized substrate is dissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The vial is vortexed for 20 seconds and left at room temperature, in the dark, with occasional mixing until dissolved. Then 900 microliters of 100 mM acetate, 0.01% (w/v) TRITON® X100, 0.12 mM CaCl2, pH 5.5 is added, vortexed thoroughly and stored at room temperature, in the dark until ready to use. The substrate working solution is prepared by diluting 10-fold in residual activity buffer (100 mM acetate, 0.01% (w/v) TRITON® X100, 0.12 mM CaCl2, pH 5.5) giving a substrate concentration of 100 micrograms/ml. Immediately after incubation the enzyme is diluted to a concentration of 20 ng enzyme protein/mL in 100 mM acetate, 0.01% (W/v) TRITON® X100, 0.12 mM CaCl2, pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixed for 10 second with 25 microliters of the diluted enzyme in a black 384 well microtiter plate. The fluorescence intensity is measured (excitation: 485 nm, emission: 555 nm) once every minute for 15 minutes in each well at 25° C. and the Vmax is calculated as the slope of the plot of fluorescence intensity against time. The plot should be linear and the residual activity assay has been adjusted so that the diluted reference enzyme solution is within the linear range of the activity assay.

Example 1 Preparation of Hybrids

The following hybrids of the calcium-sensitive alpha-amylase having the sequence shown in SEQ ID NO: 7 and the calcium-insensitive alpha-amylase having the sequence shown in SEQ ID NO: 13 were prepared.

Hybrid 1: the amino acid residues 106-215 of SEQ ID NO: 7 were removed and replaced with the amino acid residues 103-211 of SEQ ID NO: 13, which results in SEQ ID NO: 17, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 17 numbering), which correspond to E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 18.
Hybrid 2: the amino acid residues 106-214 of SEQ ID NO: 7 were removed and replaced with the amino acid residues 103-210 of SEQ ID NO: 13, which results in SEQ ID NO: 19, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 19 numbering), which correspond to E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 20.
Hybrid 3: the amino acid residues 106-213 of SEQ ID NO: 7 were removed and replaced with the amino acid residues 103-209 of SEQ ID NO: 13, which results in SEQ ID NO: 21, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 21 numbering), which correspond to E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 22.

Example 2 Stability in the Presence of Chelator

Enzyme samples were incubated in buffer pH 8.0 (50 mM EPPS, 0.01% TRITON® X100, pH 8.0) with 1.5% final concentration of DTPA at 49° C. for 1 hour and reference samples were incubated at 4° C. for 1 hour. In addition, enzyme samples were incubated in buffer pH 10.0 (50 mM EPPS, 0.01% TRITON® X100, pH 10.0) with 1.5% final concentration of DTPA at 42° C. for 1 hour and reference samples were incubated at 4° C. for 1 hour.

For the determination of amylase stability in buffer pH 8 and pH 10 with DTPA the enzymes to be tested were adjusted to 0.25 and 0.5 mg enzyme protein/mL by diluting in 5 mM EPPS, 0.01% TRITON® X100, pH 8.0.

160 microliters stability buffer (50 mM EPPS, 0.01% TRITON® X100, 1.875% DTPA, pH 8.0 or pH 10.0) and 40 microliters of the amylase solution were transferred to a 96-well PCR microtiter plate in duplicate and the content was mixed for 1 minute. Final concentration of DTPA was 1.5% in each well. 20 microliters from each well was transferred to a new PCR microtiter plate, which was placed at 4° C. (reference sample). The PCR MTP was incubated in PCR machine for 1 hour at 49° C. when buffer had pH 8.0 (pH 8, 49° C. samples) and for 1 hour at 42° C. when buffer had pH 10.0 (pH 10, 42° C. samples).

Immediately after incubation, the samples on PCR plates were analyzed for amylase activity as described in the G7-pNP Amylase assay. It should be noted that in order to reduce interference from DTPA on the assay, both reference and pH 8, 49° C. samples/pH 10, 42° C. samples were diluted to the same concentration before being analyzed for residual activity. The activity of both the reference samples and the pH 8, 49° C. samples or pH 10, 42° C. samples were determined on the same 96 well plate. The residual activity was calculated as 100*Vmax(pH 8, 42° C. or pH 10, 49° C. sample)/Vmax(reference sample).

Residual activity in % Residual activity in % after 1 hour 49° C., after 1 hour 42° C., pH 8.0 and 1.5% pH 10.0 and 1.5% Enzyme DTPA DTPA SEQ ID NO: 7 with the 20 18 deletions D183* + T184* (SEQ ID NO: 7 numbering) SEQ ID NO: 7 1 9 SP707 (SEQ ID NO: 8) 1 3 Hybrid 1 100 102 Hybrid 2 102 102 Hybrid 3 103 103 Hybrids 1, 2 and 3 are highly stable and have 100% residual activity after incubation for 1 hour at both pH 8, 49° C. and pH 10, 42° C. In comparison SEQ ID NO: 7 with the deletions D183* + T184* has less than 20% residual activity at these conditions and SEQ ID NO: 7 and SP707 have even less residual activity.

Example 3 Additional Alpha-Amylases

The following alpha-amylases were prepared:

Hybrid 4: the amino acid residues 106-212 of SEQ ID NO: 5 were removed and replaced with the amino acid residues 103-208 of SEQ ID NO: 13, which results in SEQ ID NO: 23, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 23 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 24.
Hybrid 5: the amino acid residues 106-212 of SEQ ID NO: 8 were removed and replaced with the amino acid residues 103-208 of SEQ ID NO: 13, which results in SEQ ID NO: 25, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 25 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 26.
Hybrids 4 and 5 (SEQ ID NOS: 24 and 26), a variant of SEQ ID NO: 5 with the alterations E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 5 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering, and the alpha-amylase of SEQ ID NO: 8 were incubated with DTPA as described in Example 2. The results show that hybrids 4 and 5 had almost 100% residual activity after the incubations, whereas the other alpha-amylases lost most of their activity during the incubations.

Example 4 Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase with the amino acid sequence of SEQ ID NO: 28 (a hybrid of Bacillus stearothermophilus and Bacillus circulans alpha-amylases (SEQ ID NO: 27) with the alterations E181*-G182*+E187W+N188E+D191T+D407N+D430N+P432D) and alpha-amylase variants thereof was determined by incubating the reference alpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl2 followed by residual activity determination using the EnzChek® substrate (EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes, Invitrogen, La Jolla, Calif., USA).

Purified enzyme samples were diluted to working concentrations of 0.5 and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01% TRITON® X100, 0.12 mM CaCl2, pH 5.0). Twenty microliters enzyme sample was transferred to 48-well PCR MTP and 180 microliters stability buffer (150 mM acetate, 150 mM MES, 0.01% TRITON® X100, 0.12 mM CaCl2, pH 4.5 or 5.5) was added to each well and mixed. The assay was performed using two concentrations of enzyme in duplicates. Before incubation at 75° C. or 85° C., 40 microliters was withdrawn and stored on ice as reference samples. Incubation was performed in a PCR machine for 30/45 minutes (pH 4.5 and 75° C.), 45/60 minutes (pH 5.5 and 75° C.), 5/10 minutes (pH 4.5 and 85° C.) and 10 minutes (pH 5.5 and 85° C.).

After incubation, the reference samples and samples from the PCR machine were diluted to 20 ng/ml in residual activity buffer (100 mM acetate, 0.01% TRITON® X100, 0.12 mM CaCl2, pH 5.5) and 25 microliters diluted enzyme was transferred to black 384-MTP. Residual activity was determined using the EnzChek® substrate as described in the section for the Enzchek® amylase activity assay. In brief, 25 microliters substrate working solution (100 micrograms/ml) is added to each well with diluted enzyme. Fluorescence was determined every minute for 15 minutes using excitation filter at 485-P nm and emission filter at 555 nm (fluorescence reader is Polarstar, BMG). The residual activity was normalized to control samples for each setup.

Assuming logarithmic decay the half life time (T % (min)) was calculated using the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T is the assay incubation time in minutes, and % RA is the % residual activity determined in the assay.

Using this assay setup the half life time was determined for the reference alpha-amylase and variants thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) T½ (min) T½ (min) (pH 4.5, 75° C., (pH 5.5, 75° C., (pH 4.5, 85° C., (pH 5.5, 85° C., Mutations (SEQ ID NO: 27 0.12 mM 0.12 mM 0.12 mM 0.12 mM numbering) CaCl2) CaCl2) CaCl2) CaCl2) Reference Alpha-Amylase 14 ± 2 33 ± 4 1.8 ± 0.2 4.5 ± 0.2 Reference Alpha-Amylase 49 4.2 13 with the substitutions M8L + N105D + K184A Reference Alpha-Amylase 58 4.7 15 with the substitutions A27Q + Q86S + A90S + N105D + K184A Reference Alpha-Amylase 59 6.8 28 with the substitutions S34K + N105D + K184A + S242Q Reference Alpha-Amylase 50 5.2 15 with the substitutions R52G + S53Y + N105D + K184A Reference Alpha-Amylase 91 11.3 29 with the substitutions V59A + A100G + N105D + T164V + K184A + M307L Reference Alpha-Amylase >120 14.1 39 with the substitutions V59A + N105D + Q150T + T164V + K184A + S242Q + M307L Reference Alpha-Amylase 108 9.4 24 with the substitutions T80D + N105D + T164V + K184A + M307L Reference Alpha-Amylase 51 6.3 19 with the substitutions A91L + N105D + K184A Reference Alpha-Amylase 69 7.3 25 with the substitutions A100L + N105D + T164V + K184A + Y222V + M307L Reference Alpha-Amylase 32 129 4 <15 with the substitution N105D Reference Alpha-Amylase 45 5.5 21 with the substitutions N105D + K117D + Q150T + K184A + S301K + G303R + A304D Reference Alpha-Amylase 20 3 6 with the substitutions N105D + E129V + R177L + A179E Reference Alpha-Amylase 64 5 19 with the substitutions N105D + E132D + K184A Reference Alpha-Amylase 33 3.3 9 with the substitutions N105D + F134E + K184A Reference Alpha-Amylase 44 3.9 16 with the substitutions N105D + E135N + A179N + K184A Reference Alpha-Amylase 27 117 4 10 with the substitutions N105D + Q150T Reference Alpha-Amylase 59 7.3 20 with the substitutions N105D + Q150T + T164V + F166W + A168E + E171K + K184A + N407D + N430D + D432P Reference Alpha-Amylase >120 13.6 >40 with the substitutions N105D + Q150T + T164V + K184A + S242Q + M284T + M307L Reference Alpha-Amylase 114 13.4 >40 with the substitutions N105D + Q150T + T164V + K184A + S242Q + M284T + N407D Reference Alpha-Amylase 37 141 5 13 with the substitutions N105D + Q150T + F166W + A168E + E171K Reference Alpha-Amylase 68 7.9 21 with the substitutions N105D + Q150T + F166W + A168E + E171K + K184A Reference Alpha-Amylase 46 207 5.1 15 with the substitutions N105D + Q150T + K184A Reference Alpha-Amylase 42 4.7 14 with the substitutions N105D + Q150T + K184A + Y206M Reference Alpha-Amylase 41 111 4.6 16 with the substitutions N105D + Q150T + K184A + S301K + G303R + A304D Reference Alpha-Amylase 50 5.2 15 with the substitutions N105D + N157Y + E159Y + H160Y + K184A + H208Y + D210Y Reference Alpha-Amylase 38 4.3 13 with the substitutions N105D + H160Y + K184A Reference Alpha-Amylase 71 7.5 18 with the substitutions N105D + T164V + K184A + Y222V + M307L Reference Alpha-Amylase >120 11.7 39 with the substitutions N105D + T164V + K184A + S242Q + M284T + M307L Reference Alpha-Amylase 68 5.7 19 with the substitutions N105D + T164V + K184A + F244Y + M284T + M307L Reference Alpha-Amylase 105 12 >40 with the substitutions N105D + T164V + K184A + M284Q + M307L Reference Alpha-Amylase >120 14.6 >40 with the substitutions N105D + T164V + K184A + M284V + M307L Reference Alpha-Amylase 70 230 8.7 23 with the substitutions N105D + T164V + K184A + M307L Reference Alpha-Amylase 49 140 4 13 with the substitutions N105D + F166W + A168E + E171K Reference Alpha-Amylase 33 112 4.8 14 with the substitutions N105D + F166W + A168E + E171K + S301K + G303R + A304D Reference Alpha-Amylase 68 5.8 19 with the substitutions N105D + A179D + K184A Reference Alpha-Amylase 56 5.3 >40 with the substitutions N105D + A179N + K184A Reference Alpha-Amylase 56 6 28 with the substitutions N105D + A179Q + K184A Reference Alpha-Amylase 40 153 5.6 14 with the substitutions N105D + K184A Reference Alpha-Amylase 55 5.8 15 N105D + K184A + L388V Reference Alpha-Amylase 45 5.6 17 with the substitutions N105D + K184A + N407D + N430D + D432P Reference Alpha-Amylase 52 5.5 17 with the substitutions N105D + K184A + D432P Reference Alpha-Amylase 47 5.2 18 with the substitutions N105D + K184A + T459P Reference Alpha-Amylase 8 27 4 with the substitutions N105D + Y206K Reference Alpha-Amylase 18 81 2.2 11 with the substitutions N105D + Y206M Reference Alpha-Amylase 37 4.1 11 with the substitutions N105D + K220P + N224L Reference Alpha-Amylase 23 116 2.9 10 with the substitutions N105D + S301K + G303R + A304D Reference Alpha-Amylase 23 2 6 with the substitutions V115W + F134Y + E135Q + K169S + G170R + R172L + G174R + F176Y Reference Alpha-Amylase 17 37 2 5 with the substitution K117D Reference Alpha-Amylase 9 21 with the substitutions E129V + Q150T Reference Alpha-Amylase 21 78 2.2 7 with the substitutions F134Y + E135Q + K169S + G170R + R172L + G174R + F176Y Reference Alpha-Amylase 32 90 4 10 with the substitution Q150T Reference Alpha-Amylase 21 49 3 7 with the substitution T164V Reference Alpha-Amylase 30 52 3.7 <15 with the substitution K184A Reference Alpha-Amylase with the substitutions K184A + I204L Reference Alpha-Amylase 8 with the substitutions K184A + I270L Reference Alpha-Amylase 23 74 3 9 with the substitution Y206M Reference Alpha-Amylase 8 18 3 with the substitution S242E Reference Alpha-Amylase 8 18 3 with the substitutions S301K + G303R + A304D Reference Alpha-Amylase 8 20 3 with the substitution G475K Reference Alpha-Amylase 15 with the substitution G475Q

The results demonstrate that the alpha-amylase variants have a significantly greater half-life and stability than the reference alpha-amylase.

Example 5 Production of Ethanol Using Alpha-Amylase Variants

Three small scale mashes of a Bacillus stearothermophilus alpha-amylase variant sold by Novozymes under the name LIQUOZYME SC® and two alpha-amylase variants described in Example 4 were prepared as follows: about 54 g corn ground, about 51 g tap water, and about 45 g backset were mixed in a 250 mL plastic bottle to a total slurry weight of 150 g. The pH of the corn slurry was adjusted to 4.5. The enzymes were added to the mashes at 2 micrograms of amylase per gram of dry solids. For liquefaction, the alpha-amylases were added to the bottles and the bottles were mixed thoroughly and placed into a preheated 85° C. water bath. The samples were held in the water bath for 2 hours at pH 4.5 while being shaken every 10 minutes for the first 30 minutes and every 30 minutes thereafter for the remainder of the 2 hour liquefaction. The samples were then cooled in an ice bath; pH was adjusted to 5.0, and 0.75 mL urea and 0.45 mL penicillin were added to reach final concentrations of 1000 and 3 ppm in the mashes, respectively. The samples were then subjected to simultaneous saccharification and fermentation (SSF) with Spirizyme Fuel® (a glucoamylase product sold by Novozymes).

Five gram aliquots of the mashes were transferred into pre-weighed conical centrifuge tubes, using 5 replicates per mash. SSF was then performed on these mashes in a 32° C. water bath for 54 hours using Spirizyme Fuel®. The glucoamylase dose was 0.50 AGU/g DS for all fermentations. The CO2 weight loss during SSF was measured and ethanol was quantified using HPLC after 54 hours of SSF. The average 54 hour HPLC SSF data are provided in Table 2 below.

TABLE 2 Ethanol Yields After 54 Hours Fermentation Ethanol, Alpha-Amylase g/L Std dev. LIQUOZYME SC ® 105.5946 0.3708 Reference Alpha-Amylase disclosed in 115.6339 0.5562 Example 4 with the substitution N105D Reference Alpha-Amylase disclosed in 116.7224 0.8226 Example 4 with the substitution K184A

The results demonstrate that the use of the alpha-amylase variants resulted in a significantly greater yield of ethanol relative to LIQUOZYME SC®.

Example 6 Wash Performance in a Detergent

In order to assess the wash performance of alpha-amylases in a detergent, washing experiments were performed. The performance of hybrids 1, 2 and 3 of Example 1 was tested using the Mini Wash Assay and compared to the alpha-amylase having the amino acid sequence of SEQ ID NO: 7 with the deletions D183*+T184* (SEQ ID NO: 7 numbering). In this test, the wash performance of enzyme-detergent solutions can be examined at several enzyme dosages simultaneously.

Description of the Mini Wash Assay

The Mini Wash has a number of beakers with each beaker able to hold up to 80 ml enzyme-detergent solution. Water hardness is adjusted to 10° dH by addition of CaCl2, MgCl2, and NaHCO3 to the test system. A textile sample, in this case CS-28, is attached to a frame designed to dip the textile into the enzyme-detergent solution with a frequency of 40 submersions per min. The temperature of the enzyme-detergent solution is controlled during wash. After wash the textile is rinsed in running tap water and subsequently dried in the dark. The wash performance of the enzyme-detergent solution is evaluated by measuring the remission at 460 nm with a ZEISS MCS 521 VIS Spectrophotometer.

Textiles:

CS-28 is a technical rice starch stained cotton textile that can be obtained from Center For Test materials BV, P.O. Box 120, 3133 KT Vlaardingen, the Netherlands.

The experiment was conducted under the experimental conditions specified below:

Detergent Commercial Tide 2X Ultra, inactivated by boiling for 15 minutes Detergent dosage 0.78 g/L Test solution volume 60 mL pH After wash pH was measured to 8.3 Wash time 20 minutes followed by 5 minutes rinse Temperature 40° C. Water hardness 10°dH, Ca/Mg 3:1 Enzyme concentration 0; 0.03; 0.06; 0.12; 0.20; 1.0 mg purified in test solution enzyme protein/L Enzymes SEQ ID NO: 7 with the deletions D183* + T184* Hybrid 1 Hybrid 2 Hybrid 3 Test material CS-28 (Rice starch on cotton)

Results and Discussion:

The wash performance of the alpha-amylases was normalized to the wash performance of the alpha-amylase having the amino acid sequence of SEQ ID NO: 7 with the deletions D183*+T184*.

SEQ ID NO: 7 with the Dose (mg enzyme deletions protein/L) D183* + T184* Hybrid 1 Hybrid 2 Hybrid 3 0 100.0 100.0 100.0 100.0 0.03 100.0 108.8 105.6 93.7 0.06 100.0 109.4 103.4 92.9 0.12 100.0 108.7 104.5 90.4 0.2 100.0 106.2 99.6 94.4 1 100.0 101.2 99.2 96.7

Example 7 Preparation of Hybrids

The following hybrids were prepared.

Hybrid 6: the amino acid residues 106-213 in a variant of SEQ ID NO: 5 having the substitutions M9L+R118K+ G149A+G182T+G186A+D183*+G184*+N195F+M202L+V214V+T257I+Y295F+N299Y+R320K+ M323T+A339S+E345R+R458K (using SEQ ID NO: 5 numbering) were removed and replaced with the amino acid residues 103-209 of SEQ ID NO: 13, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T, which correspond to E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 32.
Hybrid 7: the amino acid residues 106-213 in a variant of SEQ ID NO: 8 (using SEQ ID NO: 8 numbering) were removed and replaced with the amino acid residues 103-209 of SEQ ID NO: 13. The sequence of this hybrid is shown in SEQ ID NO: 33.
Hybrid 8: the amino acid residues 104-208 in a variant of SEQ ID NO: 3 having amino acids 1-35 replaced by amino acids 1-33 of SEQ ID NO: 1 and having the substitutions G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using SEQ ID NO: 3 numbering) were removed and replaced with the amino acid residues 103-209 of SEQ ID NO: 13, and the following alterations were introduced: N102D, Q147T, E178*, N179*, K181A, E184W, N185E and D188T, which correspond to N105D, Q150T, E181*, N182*, K184A, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 34.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

The invention is further defined by the following paragraphs:

Paragraph 1. An isolated alpha-amylase, comprising an A-domain of a calcium-sensitive alpha-amylase, a B-domain, and a C-domain of a calcium-sensitive alpha-amylase, wherein

(a) the B-domain has at least 55% and less than 100% sequence identity with the B-domain of SEQ ID NO: 13; and

(b) the alpha-amylase has alpha-amylase activity.

Paragraph 2. The alpha-amylase of paragraph 1, wherein the amino acid at the position corresponding to position 105 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 3. The alpha-amylase of paragraph 1 or 2, wherein the amino acid at the position corresponding to position 115 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, e.g., Trp.
Paragraph 4. The alpha-amylase of any of paragraphs 1-3, wherein the amino acid at the position corresponding to position 117 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 5. The alpha-amylase of any of paragraphs 1-4, wherein the amino acid at the position corresponding to position 129 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Val.
Paragraph 6. The alpha-amylase of any of paragraphs 1-5, wherein the amino acid at the position corresponding to position 132 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 7. The alpha-amylase of any of paragraphs 1-6, wherein the amino acid at the position corresponding to position 134 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu or Tyr.
Paragraph 8. The alpha-amylase of any of paragraphs 1-7, wherein the amino acid at the position corresponding to position 135 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn or Gln.
Paragraph 9. The alpha-amylase of any of paragraphs 1-8, wherein the amino acid at the position corresponding to position 150 is Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr.
Paragraph 10. The alpha-amylase of any of paragraphs 1-9, wherein the amino acid at the position corresponding to position 157 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 11. The alpha-amylase of any of paragraphs 1-10, wherein the amino acid at the position corresponding to position 159 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 12. The alpha-amylase of any of paragraphs 1-11, wherein the amino acid at the position corresponding to position 160 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 13. The alpha-amylase of any of paragraphs 1-12, wherein the amino acid at the position corresponding to position 164 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, e.g., Val.
Paragraph 14. The alpha-amylase of any of paragraphs 1-13, wherein the amino acid at the position corresponding to position 166 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp.
Paragraph 15. The alpha-amylase of any of paragraphs 1-14, wherein the amino acid at the position corresponding to position 168 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu.
Paragraph 16. The alpha-amylase of any of paragraphs 1-15, wherein the amino acid at the position corresponding to position 169 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ser.
Paragraph 17. The alpha-amylase of any of paragraphs 1-16, wherein the amino acid at the position corresponding to position 170 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg.
Paragraph 18. The alpha-amylase of any of paragraphs 1-17, wherein the amino acid at the position corresponding to position 171 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Lys.
Paragraph 19. The alpha-amylase of any of paragraphs 1-18, wherein the amino acid at the position corresponding to position 172 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.
Paragraph 20. The alpha-amylase of any of paragraphs 1-19, wherein the amino acid at the position corresponding to position 174 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg.
Paragraph 21. The alpha-amylase of any of paragraphs 1-20, wherein the amino acid at the position corresponding to position 176 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 22. The alpha-amylase of any of paragraphs 1-21, wherein the amino acid at the position corresponding to position 177 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.
Paragraph 23. The alpha-amylase of any of paragraphs 1-22, wherein the amino acid at the position corresponding to position 179 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn, Asp, Gln, or Glu, or is absent.
Paragraph 24. The alpha-amylase of any of paragraphs 1-23, wherein the amino acid at the position corresponding to position 180 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.
Paragraph 25. The alpha-amylase of any of paragraphs 1-24, wherein the amino acid at the position corresponding to position 181 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Asp, Cys, Leu, or Pro, or is absent.
Paragraph 26. The alpha-amylase of any of paragraphs 1-25, wherein the amino acid at the position corresponding to position 182 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.
Paragraph 27. The alpha-amylase of any of paragraphs 1-26, wherein the amino acid at the position corresponding to position 184 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala.
Paragraph 28. The alpha-amylase of any of paragraphs 1-27, wherein the amino acid at the position corresponding to position 187 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp.
Paragraph 29. The alpha-amylase of any of paragraphs 1-28, wherein the amino acid at the position corresponding to position 188 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu.
Paragraph 30. The alpha-amylase of any of paragraphs 1-29, wherein the amino acid at the position corresponding to position 191 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr.
Paragraph 31. The alpha-amylase of any of paragraphs 1-30, wherein the amino acid at the position corresponding to position 206 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, e.g., Lys or Met.
Paragraph 32. The alpha-amylase of any of paragraphs 1-31, wherein the amino acid at the position corresponding to position 208 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 33. The alpha-amylase of any of paragraphs 1-32, wherein the amino acid at the position corresponding to position 210 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr or Val.
Paragraph 34. The alpha-amylase of any of paragraphs 1-33, wherein the B-domain has at least one difference at a position corresponding to position 105, 115, 117, 129, 132, 134, 135, 150, 157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177, 179, 184, 187, 188, 191, 206, 208, or 210, and the amino acids at at least two positions corresponding to positions 179-182 are absent.
Paragraph 35. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 179 and 180 are absent.
Paragraph 36. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 179 and 181 are absent.
Paragraph 37. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 179 and 182 are absent.
Paragraph 38. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 180 and 181 are absent.
Paragraph 39. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 180 and 182 are absent.
Paragraph 40. The alpha-amylase of any of paragraphs 1-34, wherein the amino acids at the positions corresponding to positions 181 and 182 are absent.
Paragraph 41. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 13 at the positions corresponding to positions 105, 117, 150, and 184.
Paragraph 42. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 129, 177, and 179.
Paragraph 43. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 132, and 184.
Paragraph 44. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 134, and 184.
Paragraph 45. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 135, 179, and 184.
Paragraph 46. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105 and 150.
Paragraph 47. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, 164, 166, 168, 171, and 184.
Paragraph 48. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, 164, and 184.
Paragraph 49. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, 166, 168, and 171.
Paragraph 50. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, 166, 168, 171, and 184.
Paragraph 51. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, and 184.
Paragraph 52. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 150, 184, and 206.
Paragraph 53. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 157, 159, 160, 184, 208, and 210.
Paragraph 54. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 160, and 184.
Paragraph 55. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 164, and 184.
Paragraph 56. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 166, 168, and 171.
Paragraph 57. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 179, and 184.
Paragraph 58. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105 and 184.
Paragraph 59. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105, 184, and 210.
Paragraph 60. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 105 and 206.
Paragraph 61. The alpha-amylase of any of paragraphs 1-40, wherein the B-domain is different from the B-domain of SEQ ID NO: 3 at the positions corresponding to positions 187, 188, and 191.
Paragraph 62. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 1.
Paragraph 63. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 2.
Paragraph 64. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at +least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 3.
Paragraph 65. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 4.
Paragraph 66. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 5.
Paragraph 67. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 6.
Paragraph 68. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 7.
Paragraph 69. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 8.
Paragraph 70. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 9.
Paragraph 71. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 10.
Paragraph 72. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 11.
Paragraph 73. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 12.
Paragraph 74. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 29.
Paragraph 75. The alpha-amylase of any of paragraphs 1-61, wherein the A-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the A-domain of SEQ ID NO: 30.
Paragraph 76. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 91-111, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 96-101 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 101-106, in particular positions 1-101 of SEQ ID NO: 1.
Paragraph 77. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 2.
Paragraph 78. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 93-113, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 98-103 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 103-108, in particular positions 1-103 of SEQ ID NO: 3.
Paragraph 79. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 94-114, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 99-104 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109, in particular positions 1-104 of SEQ ID NO: 4.
Paragraph 80. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 5.
Paragraph 81. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 6.
Paragraph 82. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 7.
Paragraph 83. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 8.
Paragraph 84. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 9.
Paragraph 85. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 10.
Paragraph 86. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 11.
Paragraph 87. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 95-115, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 100-105 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 105-110, in particular positions 1-105 of SEQ ID NO: 12.
Paragraph 88. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 94-114, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 99-104 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109, in particular positions 1-104 of SEQ ID NO: 29.
Paragraph 89. The alpha-amylase of any of paragraphs 1-75, wherein the A1-domain has at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 1-5 and ending a position in the range of positions 92-112, e.g., starting at a position in the range of positions 1-3 and ending at a position in the range of positions 97-102 or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 102-107, in particular positions 1-102 of SEQ ID NO: 30.
Paragraph 90. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 1.
Paragraph 91. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 2.
Paragraph 92. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 3.
Paragraph 93. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 4.
Paragraph 94. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 5.
Paragraph 95. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 6.
Paragraph 96. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 7.
Paragraph 97. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 8.
Paragraph 98. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 9.
Paragraph 99. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 10.
Paragraph 100. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 11.
Paragraph 101. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 12.
Paragraph 102. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 29.
Paragraph 103. The alpha-amylase of any of paragraphs 1-89, wherein the C-domain has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the C-domain of SEQ ID NO: 30.
Paragraph 104. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 198-218 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 480-483, in particular positions 208-483 of SEQ ID NO: 1.
Paragraph 105. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 202-222 and ending at a position in the range of positions 479-484, e.g., starting at a position in the range of positions 207-212 and ending at a position in the range of positions 481-484 or starting at a position in the range of positions 212-217 and ending at a position in the range of positions 481-484, in particular positions 212-484 of SEQ ID NO: 2.
Paragraph 106. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 198-218 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 203-208 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 208-213 and ending at a position in the range of positions 480-483, in particular positions 208-483 of SEQ ID NO: 3.
Paragraph 107. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 201-221 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 206-211 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 211-216 and ending at a position in the range of positions 480-483, in particular positions 211-483 of SEQ ID NO: 4.
Paragraph 108. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 193-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 5.
Paragraph 109. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 6.
Paragraph 110. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 7.
Paragraph 111. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 8.
Paragraph 112. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 481-484, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-484 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-484, in particular positions 213-484 of SEQ ID NO: 9.
Paragraph 113. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-484, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-484 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-484, in particular positions 213-484 of SEQ ID NO: 10.
Paragraph 114. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 11.
Paragraph 115. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 203-223 and ending at a position in the range of positions 482-485, e.g., starting at a position in the range of positions 208-213 and ending at a position in the range of positions 482-485 or starting at a position in the range of positions 213-218 and ending at a position in the range of positions 482-485, in particular positions 213-485 of SEQ ID NO: 12.
Paragraph 116. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 201-221 and ending at a position in the range of positions 478-483, e.g., starting at a position in the range of positions 206-211 and ending at a position in the range of positions 480-483 or starting at a position in the range of positions 211-216 and ending at a position in the range of positions 480-483, in particular positions 211-483 of SEQ ID NO: 29.
Paragraph 117. The alpha-amylase of any of paragraphs 1-103, wherein the A2 and C-domains have at least 60% sequence identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence starting at a position in the range of positions 199-219 and ending at a position in the range of positions 479-484, e.g., starting at a position in the range of positions 204-209 and ending at a position in the range of positions 481-484 or starting at a position in the range of positions 209-214 and ending at a position in the range of positions 481-484, in particular positions 209-484 of SEQ ID NO: 30.
Paragraph 118. The alpha-amylase of any of paragraphs 1-117, which is more thermostable than the alpha-amylase of any of SEQ ID NOS: 1-12, 29 and 30.
Paragraph 119. The alpha-amylase of any of paragraphs 1-118, which has reduced calcium sensitivity than the alpha-amylase of any of SEQ ID NOS: 1-12, 29 and 30.
Paragraph 120. The alpha-amylase of any of paragraphs 1-119, wherein the B-domain has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with the B-domain of SEQ ID NO: 13.
Paragraph 121. The alpha-amylase of any of paragraphs 1-120, wherein the B-domain has less than 99%, e.g., less than 98%, less than 97%, less than 96%, or less than 95%, sequence identity with the B-domain of SEQ ID NO: 13.
Paragraph 122. The alpha-amylase of any of paragraphs 1-121, wherein the alpha-amylase consists of 481 to 515, 481 to 493, or 481 to 486 amino acids.
Paragraph 123. A detergent composition comprising an alpha-amylase of any of paragraphs 1-122 and a surfactant.
Paragraph 124. A composition comprising an alpha-amylase of any of paragraphs 1-122 and one or more enzymes selected from the group consisting of beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase, and endoglucanase) glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, and pullulanase.
Paragraph 125. Use of an alpha-amylase of any of paragraphs 1-122 for washing and/or dishwashing.
Paragraph 126. Use of an alpha-amylase of any of paragraphs 1-122 for desizing a textile.
Paragraph 127. Use of an alpha-amylase of any of paragraphs 1-122 for producing a baked product.
Paragraph 128. Use of an alpha-amylase of any of paragraphs 1-122 for liquefying a starch-containing material.
Paragraph 129. A method of producing liquefied starch, comprising liquefying a starch-containing material with an alpha-amylase of any of paragraphs 1-122.
Paragraph 130. A process of producing a fermentation product, comprising

a. liquefying a starch-containing material with an alpha-amylase of any of paragraphs 1-122 to produce a liquefied mash;

b. saccharifying the liquefied mash to produce fermentable sugars; and

c. fermenting the fermentable sugars in the presence of a fermenting organism.

Paragraph 131. The process of paragraph 130, wherein the starch-containing material is liquefied with the alpha-amylase and a pullulanase, e.g., a GH57 pullulanase.
Paragraph 132. The process of paragraph 131, wherein the pullulanase is obtained from a strain of Thermococcus, including Thermococcus sp. AM4, Thermococcus sp. HJ21, Thermococcus barophilus, Thermococcus gammatolerans, Thermococcus hydrothermalis; Thermococcus kodakarensis, Thermococcus litoralis, and Thermococcus onnurineus; or from a strain of Pyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus.
Paragraph 133. The process of any of paragraphs 130-132, further comprising adding a protease, such as an acid fungal protease or a metalloprotease before, during and/or after liquefaction.
Paragraph 134. The process of paragraph 133, wherein the metalloprotease is obtained from a strain of Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.
Paragraph 135. A process of producing a fermentation product, comprising contacting a starch substrate with alpha-amylase of any of paragraphs 1-122, a glucoamylase, and a fermenting organism.
Paragraph 136. The process of any of paragraphs 130-135, wherein the fermentation product is selected from the group consisting of alcohol (e.g., ethanol and butanol), organic acids (e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).
Paragraph 137. An isolated polynucleotide encoding the alpha-amylase of any of paragraphs 1-122.
Paragraph 138. A nucleic acid construct comprising the polynucleotide of paragraph 137. Paragraph 139. An expression vector comprising the nucleic acid construct of paragraph 138.
Paragraph 140. A host cell comprising the nucleic acid construct of paragraph 138.
Paragraph 141. A method of producing an alpha-amylase, comprising:

a. cultivating the host cell of paragraph 140 under conditions suitable for the expression of the alpha-amylase; and

b. recovering the alpha-amylase from the cultivation medium.

Paragraph 142. A transgenic plant, plant part or plant cell transformed with the polynucleotide of paragraph 137.

1-19. (canceled) 20. An isolated alpha-amylase, comprising an A-domain of a calcium-sensitive alpha-amylase, a B-domain, and a C-domain of a calcium-sensitive alpha-amylase, wherein (a) the B-domain has at least 55% and less than 100% sequence identity with the B-domain of SEQ ID NO: 13; and (b) the alpha-amylase has alpha-amylase activity. 21. The alpha-amylase of claim 1, wherein the amino acid at the position corresponding to position 105 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp; the amino acid at the position corresponding to position 115 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, e.g., Trp; the amino acid at the position corresponding to position 117 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp; the amino acid at the position corresponding to position 129 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Val; the amino acid at the position corresponding to position 132 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp; the amino acid at the position corresponding to position 134 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu or Tyr; the amino acid at the position corresponding to position 135 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn or Gln; the amino acid at the position corresponding to position 150 is Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr; the amino acid at the position corresponding to position 157 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr; the amino acid at the position corresponding to position 159 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr; the amino acid at the position corresponding to position 160 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr; the amino acid at the position corresponding to position 164 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, e.g., Val; the amino acid at the position corresponding to position 166 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp; the amino acid at the position corresponding to position 168 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu; the amino acid at the position corresponding to position 169 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ser; the amino acid at the position corresponding to position 170 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg; the amino acid at the position corresponding to position 171 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Lys; the amino acid at the position corresponding to position 172 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu; the amino acid at the position corresponding to position 174 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg; the amino acid at the position corresponding to position 176 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr; the amino acid at the position corresponding to position 177 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu; the amino acid at the position corresponding to position 179 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn, Asp, Gln, or Glu, or is absent; the amino acid at the position corresponding to position 180 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent; the amino acid at the position corresponding to position 181 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Asp, Cys, Leu, or Pro, or is absent; the amino acid at the position corresponding to position 182 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent; the amino acid at the position corresponding to position 184 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala; the amino acid at the position corresponding to position 187 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp; the amino acid at the position corresponding to position 188 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu; the amino acid at the position corresponding to position 191 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr; the amino acid at the position corresponding to position 206 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, e.g., Lys or Met; the amino acid at the position corresponding to position 208 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr; and/or the amino acid at the position corresponding to position 210 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr or Val. 22. The alpha-amylase of claim 20, which is more thermostable than the alpha-amylase of any of SEQ ID NOS: 1-12, 29 and 30. 23. The alpha-amylase of claim 20, which has reduced calcium sensitivity than the alpha-amylase of any of SEQ ID NOS: 1-12, 29 and 30. 24. The alpha-amylase of claim 20, wherein the alpha-amylase consists of 481 to 515, 481 to 493, or 481 to 486 amino acids. 25. A detergent composition comprising an alpha-amylase of claim 20 and a surfactant. 26. A composition comprising an alpha-amylase of claim 20 and one or more enzymes selected from the group consisting of beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase, and endoglucanase) glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, and pullulanase. 27. A method of producing liquefied starch, comprising liquefying a starch-containing material with an alpha-amylase of claim 20. 28. A process of producing a fermentation product, comprising (a) liquefying a starch-containing material with an alpha-amylase of claim 20 to produce a liquefied mash; (b) saccharifying the liquefied mash to produce fermentable sugars; and (c) fermenting the fermentable sugars in the presence of a fermenting organism. 29. A process of producing a fermentation product, comprising contacting a starch substrate with alpha-amylase of claim 20, a glucoamylase, and a fermenting organism. 30. An isolated polynucleotide encoding the alpha-amylase of claim 20. 31. A nucleic acid construct comprising the polynucleotide of claim 30. 32. An expression vector comprising the nucleic acid construct of claim 31. 33. A host cell comprising the nucleic acid construct of claim 31. 34. A method of producing an alpha-amylase, comprising: (a) cultivating the host cell of claim 33 under conditions suitable for the expression of the alpha-amylase; and (b) recovering the alpha-amylase from the cultivation medium.


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stats Patent Info
Application #
US 20120270267 A1
Publish Date
10/25/2012
Document #
File Date
11/29/2014
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Alpha-amylase


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