| In vivo site-specific incorporation of n-acetyl-galactosamine amino acids in eubacteria -> Monitor Keywords |
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In vivo site-specific incorporation of n-acetyl-galactosamine amino acids in eubacteriaRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Enzymatic Production Of A Protein Or Polypeptide (e.g., Enzymatic Hydrolysis, Etc.)In vivo site-specific incorporation of n-acetyl-galactosamine amino acids in eubacteria description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080064059, In vivo site-specific incorporation of n-acetyl-galactosamine amino acids in eubacteria. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and benefit of U.S. Provisional Patent Application Ser. No. 60/620,898, filed Oct. 20, 2004, the specification of which is incorporated herein in its entirety for all purposes. FIELD OF THE INVENTION [0003] The invention is in the field of protein biochemistry. The invention relates to compositions and methods for making and using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and pairs thereof, that incorporate unnatural amino acids into proteins, where the unnatural amino acids comprise an N-acetylgalactosamine moiety and the resulting proteins are glycoproteins. The invention also relates to methods of producing proteins in cells using such pairs and related compositions. The invention is in the field of glycopeptides, glycoproteins, and related mimetics, and methods for synthesis of glycopeptides, glycoproteins, and related mimetics. BACKGROUND OF THE INVENTION [0004] Glycosylation is one of most prevalent posttranslational modifications (PTMs) of proteins and plays an important role in many biological processes. For example, the posttranslational modification of proteins by glycosylation can affect protein folding and stability, modify the intrinsic activity of proteins, and modulate their interactions with other biomolecules. See, e.g., Varki, A. (1993) Glycobiology 3:97-130; Dwek (1996) Chem. Rev. 96:683-720; and Sears and Wong (1998) Cell Mol. Life. Sci. 54:223-252. Natural glycoproteins are often present as a population of many different glycoforms, which makes analysis of glycan structure and the study of glycosylation effects on protein structure and function difficult. Therefore, methods for the synthesis of natural and unnatural homogeneously glycosylated proteins would be useful tools, e.g., for the systematic understanding of glycan function, and for the development of improved glycoprotein therapeutics. [0005] Considerable effort has focused on the methods for generation of glycoproteins, including chemical and enzymatic synthetic approaches, in vitro translation, and pathway engineering. One previously known approach for making proteins having desired glycosylation patterns makes use of glycosidases to convert a heterogeneous natural glycoprotein to a simple homogenous core, onto which saccharides can then be grafted sequentially with glycosyltransferases. See, e.g., Witte, K., et al., (1997) J. Am. Chem. Soc. 119:2114-2118. A limitation of this approach is that the primary glycosylation sites are predetermined by the cell line in which the protein is expressed. Alternatively, a glycopeptide containing the desired glycan structure can be synthesized by solid phase peptide synthesis. This glycopeptide can be coupled to other peptides or recombinant protein fragments to afford a larger glycoprotein by native chemical ligation (see, e.g., Shin, Y., et al., (1999) J. Am. Chem. Soc. 121:11684-11689) expressed protein ligation, (see, e.g., Tolbert, T. J. and Wong, C.-H. (2000) J. Am. Chem. Soc. 122:5421-5428), or with engineered proteases. See, e.g., Witte, K., et al., (1998) J. Am. Chem. Soc. 120:1979-1989. Both native chemical ligation and expressed protein ligation are most effective with small proteins, and necessitate a cysteine residue at the N-terminus of the glycopeptide. When a protease is used to ligate peptides together, the ligation site is preferably placed far away from the glycosylation site for good coupling yields. See, e.g., Witte, K., et al., (1998) J. Am. Chem. Soc. 120:1979-1989. A third approach is to modify proteins with saccharides directly using chemical methods. Good selectivity can be achieved with haloacetamide saccharide derivatives, which are coupled to the thiol group of cysteine, (see, e.g., Davis, N. J. and, Flitsch, S. L. (1991) Tetrahedron Lett. 32:6793-6796; and, Macmillan, D.; et al., (2002) Org Lett 4:1467-1470), but this method can become problematic with proteins that have more than one cysteine residue. [0006] Accordingly, a need exists for improved methods for making glycoproteins having a desired glycosylation pattern. The invention fulfills this and other needs, as will be apparent upon review of the following disclosure. SUMMARY OF THE INVENTION [0007] The present invention provides methods and related compositions for the preparation of glycoproteins. In one aspect, the present invention provides orthogonal aminoacyl-tRNA synthetases that preferentially aminoacylate an orthogonal tRNA (O-tRNA) with an unnatural amino acid bearing an N-acetylgalactosamine (GalNAc) moiety, such as N-acetylgalactosamine-.alpha.-threonine or N-acetylgalactosamine-.alpha.-serine. In some embodiments, the orthogonal aminoacyl-tRNA synthetase (O-RS) is generated from a synthetase sequence originally obtained from Methanococcus jannaschii, for example, an amino acid sequence derived from a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase. Exemplary mutant M. jannaschii Tyr-tRNA synthetases (MjTyrRS) that can utilize GalNAc-containing unnatural amino acids include, but are not limited to, the polypeptide sequences provided in SEQ ID NO.: 1 through 4, as well as conservative variations thereof. [0008] In some embodiments, the O-RS aminoacylates the O-tRNA with the unnatural amino acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising the unnatural amino acid, the O-tRNA and an O-RS having an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4 or a conservative variant thereof. [0009] Also provided are nucleic acids that encode the O-RS polypeptides, for example, the nucleic acids of SEQ ID NOs: 6-9 encoding exemplary orthogonal tRNA synthetases of the present invention. Furthermore, vectors, for example expression vectors, carrying these DNA sequences are provided, as well as cells comprising the vectors. [0010] In a related aspect, the present invention provides amino acid sequences for an orthogonal aminoacyl-tRNA synthetase (O-RS) having at least 70% sequence identity with M. jannaschii Tyr-tRNA synthetase (SEQ ID NO:5), as determined using BLAST set to default parameters; the amino acid sequence include, but are not limited to, one or more amino acid substitutions (as compared to SEQ ID NO: 5) selected from the group consisting of Tyr32Phe, Tyr32Gln, Tyr32Ala, Tyr32Leu, Ala67Pro, Ala67Ser, Ala67Thr, His70Pro, His70Lys, Leu98Ile, Glu107Pro, Val149Ile, Gln155Ser, Asp158Val, Asp158Cys, Ile159Tyr, Leu162Arg, Gly163Cys, and Ala167Val. [0011] Other aspects of the invention include methods for synthesis of a glycoprotein by incorporating at a selected position into a protein an unnatural amino acid that comprises a saccharide moiety (e.g., a glycosyl-containing amino acid). A glycoprotein produced by the method is also a feature of the invention. The methods include, but are not limited to, the steps of (a) providing a translation system comprising i) an unnatural amino acid with a GalNAc moiety; ii) a nucleic acid that comprises at least one selector codon and encodes the glycoprotein; iii) an orthogonal tRNA (O-tRNA) that recognizes the selector codon; and, iv) an orthogonal aminoacyl-tRNA synthetase that preferentially aminoacylates the O-tRNA with the unnatural amino acid; and (b) incorporating the glycosylated amino acid into the selected position in the protein during translation of the protein, where the selected position in the protein corresponds to the position of the selector codon in the nucleic acid, thereby producing the protein. [0012] In some embodiments, the O-RS used in the method preferentially aminoacylates the O-tRNA with the unnatural amino acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising the glycosylated amino acid, the O-tRNA and an O-RS comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4 and conservative variants thereof. [0013] Unnatural amino acids recognized by the synthetases of the present invention include, but are not limited to, .alpha.-GalNAc-L-threonine, .alpha.-O-GalNAc-L-serine, .beta.-O-GlcNAc-L-serine, C-glycosides and thio-glycosyl analogs of a glycosyl-containing amino acid (e.g., compositions in which a carbon or a sulfur atom replaces the anomeric oxygen), and/or the like. The unnatural amino acids that can be used in the method also include N-acetylgalactosamine-.alpha.-threonine, 3,4,6-triacetyl-N-acetylgalactosamine-.alpha.-threonine, N-acetylgalactosamine-.alpha.-serine and 3,4,6-triacetyl-N-acetylgalactosamine-.alpha.-serine. Optionally, the incorporating step is performed in vivo. [0014] In some embodiments, the translation system uses an O-RS derived from a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase. [0015] The orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase perform as an orthogonal pair (O-tRNA/O-RS pair), wherein the O-tRNA recognizes the selector codon, and incorporates the unnatural amino acid (in this case, the glycosyl-containing amino acid) into the protein in response to the selector codon. Optionally, the O-tRNA employed in the methods comprises a mutRNA.sub.CUA.sup.Tyr. [0016] Exemplary orthogonal aminoacyl-tRNA synthetases for use in the incorporation of a galactosamine-containing amino acid into a peptide sequence include, but are not limited to, the polypeptides provided in SEQ ID NOS: 1-4, as well as conservative variations thereof, or is encoded by a polynucleotide comprising a polynucleotide sequence of any one of SEQ ID NOS.: 6-9 (or conservative variations thereof). Additional O-RS sequences of the present invention (e.g., that recognizes one or more glucosamine-containing amino acids) include an amino acid sequence comprising any one of SEQ ID NOS.: 11-13, or is encoded by a polynucleotide comprising a polynucleotide sequence of any one of SEQ ID NOS.: 14-16. [0017] The glycoprotein synthesis methods of the present invention can further involve contacting the saccharide moiety of the unnatural amino acid (or of the nascent polypeptide containing the incorporated unnatural amino acid) with a glycosyltransferase, a sugar donor moiety, and other reactants required for glycosyltransferase activity for a sufficient time and under appropriate conditions to transfer a sugar from the sugar donor moiety to the saccharide moiety. The product of this reaction can, if desired, be contacted by one or more additional glycosyltransferases, together with the appropriate sugar donor moieties, to further extend the saccharide component. [0018] In some embodiments of the methods, providing the translation system comprises providing a cell (e.g., a prokaryotic cell, such as a E. coli cell) in which the O-tRNA and the O-RS is expressed and/or functions. Providing the translation system can be achieved, e.g., by growing the cell in growth media, and may optionally include co-expressing a nucleic acid sequence encoding the O-RS and/or a nucleic acid encoding the O-tRNA in the cell. [0019] Optionally, providing the glycosylated amino acid involves converting a protected form of the unnatural amino acid (e.g., an acetylated version such as 3,4,6-triacetyl-N-acetylglactosamine-.alpha.-threonine) to the unprotected form (e.g., an N-acetylgalactosamine-.alpha.-threonine). Deprotection of the unnatural amino acid, e.g., by nonspecific esterases in the cytosol of the cell (or other cellular-based translation system), preferably occurs prior to aminoacylating the O-tRNA and/or incorporating the unnatural amino acid into the protein. [0020] In certain embodiments, the method further comprises contacting the product of the glycosyltransferase reaction with at least a second glycosyltransferase and a second sugar donor moiety. In one embodiment, the saccharide moiety comprises a terminal GlcNAc, the sugar donor moiety is UDP-GlcNAc and the glycosyltransferase is a .beta.3-4N-acetylglucosaminyltransferase. In another embodiment, the saccharide moiety comprises a terminal GlcNAc, the sugar donor moiety is UDP-Gal and the glycosyltransferase is a .beta.1-4-galactosyltransferase. In a further embodiment, the saccharide moiety comprises a terminal GalNAc, the sugar donor is a UDP-GlcNAc and/or a UDP-Gal, and the glycosyltransferases include a (.beta.1-6)N-acetylglucosaminyltransferase and/or a .beta.1-3-galactosyltransferase, respectively. Various sugars can optionally be added to the saccharide portion of the unnatural amino acid by using an appropriate glycosyltransferase, including, but not limited to, e.g., a galactosyltransferase, a fucosyltransferase, a glucosyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylglucosaminyltransferase, a glucuronyltransferase, a sialyltransferase, a mannosyltransferase, a glucuronic acid transferase, a galacturonic acid transferase, an oligosaccharyltransferase, and the like. [0021] In a further aspect, the present invention also provides translation systems for the preparation of glycoproteins. The translation systems of the present invention include, but are not limited to, an orthogonal tRNA (O-tRNA) that recognizes at least one selector codon, and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with an unnatural amino acid that comprises a saccharide moiety (e.g., an N-acetylgalactosamine moiety). One or more saccharide-containing unnatural amino acids are also provided, as either a protected or deprotected composition (e.g., a .beta.-O-GlcNAc-L-serine, a tri-acetyl-.beta.-O-GlcNAc-serine, an .alpha.-O-GalNAc-L-threonine, a tri-acetyl-.alpha.-O-GalNAc-threonine, an .alpha.-O-GalNAc-serine, a triacetyl-.alpha.-GalNAc-serine, the corresponding protected or unprotected thio-glycosyl analogs, and/or the like). In some embodiments, the O-RS used in the translation system is derived from a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase. In some embodiments, the translation system uses an O-tRNA that is an amber suppressor tRNA. Continue reading about In vivo site-specific incorporation of n-acetyl-galactosamine amino acids in eubacteria... 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