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Kex2 cleavage regions of recombinant fusion proteinsKex2 cleavage regions of recombinant fusion proteins description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080026376, Kex2 cleavage regions of recombinant fusion proteins. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0002]The present invention relates to increased secretion and cleavage of desired proteins, such as functional antibody proteins and industrial enzymes from filamentous fungi. The invention discloses fusion DNA constructs, vectors and fusion polypeptides incorporating KEX2 regions for protein cleavage and methods of producing desired proteins. BACKGROUND [0003]During protein secretion in a fungal cell, certain proteins are cleaved by KEX2, a member of the KEX2 or "kexin" family of serine peptidase (EC 3.4.21.61). KEX2 is a highly specific calcium-dependent endopeptidase that cleaves the peptide bond that is immediately C-terminal to a pair of basic amino acids (i.e., the "KEX2 site") in a protein substrate during secretion of that protein. KEX2 proteins generally contain a cysteine residue near the histidine residue of its active site and are inhibited by p-mercuribenzoate. The founding member of this group, the KEX2 peptidase of S. cerevisiae (Fuller et al., 1989, Proc. Natl. Acad. Sci. USA 86:1434-1438), cleaves the .alpha.-factor pheromone and killer toxin precursors during their secretion. [0004]Production of fusion polypeptides has been reported in a number of organisms including E. coli, yeast and filamentous fungi. For example, bovine chymosin has been produced in Aspergillus niger as a fusion to full length glucoamylase (GAI) (Ward et al., (1990) Bio/technology 8:435-440; U.S. Pat. No. 6,265,204 and U.S. Pat. No. 6,590,078); human interleukin 6 (hIL6) has been produced in Aspergillus nidulans as a fusion to full-length A. niger glucoamylase (GAI) (Contreras et al., (1991) Biotechnology 9:378-381); hen egg white lysozyme (Jeenes et al., (1993) FEMS Microbiol. Lett. 107:267-273) and human lactoferrin (Ward et al., (1995) Bio/Technology 13:498-503) have been produced in Aspergillus niger as a fusion to residues 1-498 of glucoamylase; and bovine chymosin has been produced in Aspergillus niger as a fusion with full length native alpha amylase (Korman et al., (1990) Curr. Genet. 17: 203-212) and in Aspergillus oryzae as a fusion with truncated forms of A. oryzae glucoamylase (Tsuchiya et al., (1994) Biosci. Biotech. Biochem. 58: 895-899). Reference is also made to Shoemaker et al., 1981 Bio/Technology 1: 691-696; Nunberg et al., (1984) Mol. Cell. Biol. 4:2306-2315 and Boel et al., (1984) EMBO J. 3:1097-1102. In some of these fusion proteins, a KEX2 protease recognition site (Lys-Arg) has been inserted between a glucoamylase and a desired protein (e.g., Contreras et al., 1991 and Ward et al., 1995). The inventors of the present invention have found that protein secretion and/or protein cleavage may be enhanced in a fusion protein when the KEX2 recognition site has been manipulated to include an amino acid KEX2 site pre-sequence. [0005]Specific literature of interest includes: Ward et al., (2004) Appl. Environ. Microbiol. 70:2567-2576; Goller et al., (1998) Appl. Environ. Microbiol. 64:3202-3208; La Grange et al., (1996) Appl. Environ. Microbiol. 62:1036-1044; Bergquist et al., (2002) Biochem. Biotechnol. 100:165-176; Spencer et al., (1998) Eur. J. Biochem. 258:107-112; Jalving et al., (2000) Appl. Environ. Microbiol. 66:363-368); Brenner and Fuller (1992) Proc. Natl. Acad. Sci. 89:922-926; Durand et al., (1999) Appl. Microbiol. Biotechnol. 52: 208-214; Ahn et al., (2004) Appl. Microbiol. Biotechnol. 64:833-839; Gouka et al., (1997) Appl Microbiol Biotechnol. 47:1-11 Broekhuijsen et al., (1993) J. Biotechnol. 31:135-145; MacKenzie et al., (1998) J. Biotechnol. 63:137-146 and published patent applications 20040018573 and 20050158825. Also U.S. Pat. No. 4,816,567 and U.S. Pat. No. 6,331,415 disclose processes for producing immunoglobulin molecules in recombinant host cells. The above cited literature is incorporated by reference herein for all purposes. [0006]While numerous methods are available for the production of industrial enzymes and therapeutic proteins, there remains a need for alternative methods of protein production and particularly for therapeutic protein production, such as antibody production, which will result in relatively quick scale up time and high levels of produced protein with limited risk of contamination by viral or other adventitious agents. The present invention answers this need. SUMMARY OF THE INVENTION [0007]A fusion DNA construct, vectors, a fusion polypeptide, a cell comprising the fusion DNA construct, and methods for enhancing the secretion and/or cleavage of a desired protein from a cell are provided. More specifically, a KEX2 region encompassed by the invention has been included in a fusion polypeptide to provide for cleavage of a desired protein from the fusion polypeptide. Accordingly, the invention pertains to a KEX2 region for protein cleavage. [0008]In some embodiments, the invention relates to a fusion DNA construct encoding a fusion polypeptide, comprising in operable linkage from the 5' end of said construct, a promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; a third DNA molecule encoding a KEX2 region, said KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5' to the KEX2 site; and a fourth DNA molecule encoding a desired protein. In some aspects of this embodiment, the invention relates to a vector, such as an expression vector, which comprises the fusion DNA construct, and in other aspects the invention relates to host cells transformed with the vector or comprising the fusion DNA construct. [0009]In other embodiments, the invention relates to a fusion polypeptide comprising from an amino terminus of said fusion polypeptide a first amino acid sequence comprising a signal sequence functional as a secretory sequence; a second amino acid sequence comprising a carrier protein; a third amino acid sequence comprising a KEX2 region, said KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately N-terminal to said KEX2 site; and a fourth amino acid sequence comprising a desired protein. [0010]In further embodiments, the invention relates to a KEX2 region (X.sub.4X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2) comprising a KEX2 site (B.sub.1B.sub.2) and a KEX2 site pre-sequence (X.sub.4X.sub.3X.sub.2X.sub.1) immediately N-terminal to said KEX2 site. [0011]In yet other embodiments, the invention relates to a process of producing a desired protein in a filamentous fungal host cell and particularly in a Trichoderma cell, comprising obtaining a filamentous fungal host cell comprising a fusion DNA construct according to the invention and culturing the filamentous fungal host cell under suitable conditions which allow the expression and secretion of the desired protein. In some aspects of this embodiment, the desired protein will be recovered. In other aspects of this embodiment, the cleavage of the desired protein from the fusion polypeptide will be greater than the cleavage of the same desired protein from an equivalent fusion polypeptide that lacks the KEX2 site pre-sequence. In other aspects of this embodiment, the secretion of the desired protein from the fusion polypeptide will be greater than the secretion of the same desired protein from an equivalent fusion polypeptide, which lacks the KEX2 site pre-sequence. [0012]In an additional embodiment, the invention relates to a method for identifying enhanced secretion and/or cleavage of a desired protein comprising a) altering a KEX2 site pre-sequence of a parental fusion polypeptide, said parental fusion polypeptide comprising a signal sequence; a KEX2 region comprising a KEX2 site and the KEX2 site pre-sequence which is located immediately N-terminal to said KEX2 site, and an amino acid sequence comprising a desired protein to produce a set of test fusion polypeptides that are identical to said parental fusion polypeptide except for said KEX2 site pre-sequence; b) evaluating secretion of said test fusion polypeptides and said parental fusion polypeptide by a filamentous fungal cell; c) identifying a test fusion polypeptide that has enhanced secretion and/or cleavage as compared to said parental fusion polypeptide. [0013]In further aspects of this embodiment, the invention relates to a method of identifying an optimized KEX2 site pre-sequence which comprises, testing a plurality of different test fusion polypeptides obtained as described above; and determining which of said different test fusion polypeptides has greater secretion and/or protein cleavage, wherein said optimized KEX2 site pre-sequence is the altered KEX2 site pre-sequence of the test fusion polypeptide that has the greatest secretion and/or protein cleavage. BRIEF DESCRIPTION OF THE FIGURES [0014]Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures: [0015]FIG. 1 schematically illustrates an embodiment of a fusion polypeptide according to the invention, including a carrier protein, a KEX2 region and a desired protein, wherein the carrier protein is illustrated as a cellobiohydrolase I (CBH1) core/linker, which comprises the catalytic domain and part of the linker region of the CBH1 protein and the desired protein is illustrated as an antibody light chain or heavy chain. [0016]FIG. 2 depicts a map of the pTrex4-her2 light chain DNA2.0 plasmid used for the expression of a fusion polypeptide. The plasmid includes a Trichoderma reesei cbh1 promoter; a polynucleotide encoding a CBH1 signal sequence and carrier protein; a KEX2 region inserted immediately after the SpeI site, a polynucleotide encoding the desired protein illustrated as an antibody (trastuzumab) light chain; a Trichoderma reesei cellobiohydrolase (cbh1) terminator; an amdS Aspergillus nidulans acetamidase marker. [0017]FIG. 3A-E provide the nucleotide sequence (SEQ ID NO: 103) (10885 bp) of the pTrex4-her2 light chain DNA2.0 plasmid of FIG. 2. [0018]FIG. 4 shows a Western blot of supernatants of cultured Trichoderma reesei cells comprising KEX2 region sequences as further described in examples 1, 2, 3 and 4. Lane 1 represents a molecular weight marker (See Blue Plus 2, Invitrogen). Lanes 2 and 3 represent a GGGKR variant (SEQ ID NO: 5); lane 4 represents a GGGKRGGG variant (SEQ ID NO: 7); lane 5 represents a VAVEKR variant (SEQ ID NO: 9) KEX2 region encompassed by the invention; and lanes 6 and 7 represent a KRGGG variant (SEQ ID NO: 2). [0019]FIG. 5 shows a Western blot of supernatants of cultured Trichoderma reesei cells comprising KEX2 regions encompassed by the invention as further described in example 5. Lane 1 represents a molecular weight marker as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 correspondingly represent VAVEKR (SEQ ID NO: 9), VAVWKR (SEQ ID NO: 25), VAVGKR (SEQ ID NO: 26), VAVRKR (SEQ ID NO: 27), VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVAKR (SEQ ID NO: 30), VAVLKR (SEQ ID NO: 31), VAVDKR (SEQ ID NO: 32), VAVNKR (SEQ ID NO: 33), VAVYKR (SEQ ID NO: 34), VAVHKR (SEQ ID NO: 35) KEX2 regions. [0020]FIG. 6 shows a Western blot of supernatants of cultured Trichoderma reesei cells containing KEX2 region sequences as further described in examples 5, 6, and 7. Lanes 1 and 10 represent a molecular weight marker, as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 and 16 correspondingly represent AAVEKR (SEQ ID NO: 38), GAVEKR (SEQ ID NO: 37), MAVEKR (SEQ ID NO: 36), LAVEKR (SEQ ID NO: 39), WAVEKR (SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), DAVEKR (SEQ ID NO: 51), VAVEKR (SEQ ID NO: 9), HAVEKR (SEQ ID NO: 52), QAVEKR (SEQ ID NO: 47), SAVEKR (SEQ ID NO: 46), NVISKR (SEQ ID NO: 22), and SDVTKR (SEQ ID NO: 24) KEX2 regions. [0021]FIG. 7 shows a Western blot of supernatants of cultured Trichoderma reesei cells containing KEX2 region sequences as further described in example 5. Lane 1 represents a molecular weight marker, as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 correspondingly represent VAVEKR (SEQ ID NO: 9), VGVEKR (SEQ ID NO: 56), VTVEKR (SEQ ID NO: 65), VEVEKR (SEQ ID NO: 55), VPVEKR (SEQ ID NO: 62), VWVEKR (SEQ ID NO: 67), VKVEKR (SEQ ID NO: 58), VRVEKR (SEQ ID NO: 63), VVVEKR (SEQ ID NO: 66), and VIVEKR (SEQ ID NO: 57) KEX2 regions. 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