Patent ID: 8637306

Claim:
A biologically and/or pharmacologically active carrier polypeptide-target polypeptide conjugate for therapeutic use, produced by a method comprising: incorporating a first unnatural amino acid residue comprising a first reactive group into a carrier polypeptide during translation of the carrier polypeptide in a methylotrophic yeast cell; incorporating a second unnatural amino acid residue comprising a second reactive group into a therapeutic target polypeptide during synthesis or translation of the target polypeptide, wherein the target polypeptide is different from the carrier polypeptide, wherein the first and second reactive groups are different and are selected from a keto group and an aminooxy group; wherein incorporating the first unnatural amino acid into the carrier polypeptide during translation comprises: (a) providing a methylotrophic yeast cell comprising (i) the first unnatural amino acid, (ii) an orthogonal tRNA-synthetase (O-RS) derived from Escherichia coli , wherein providing an O-RS comprises providing an O-RS polynucleotide under the transcriptional control of a FLD 1 promoter; (iii) an orthogonal tRNA (O-tRNA) derived from Escherichia coli , wherein the O-RS and the O-tRNA are encoded in a cassette on a single plasmid and stably integrated into the cell genome at an ARG4 gene, and wherein the O-RS preferentially aminoacylates the O-tRNA with the first unnatural amino acid in the methylotrophic yeast cell; and (iv) a nucleic acid encoding the carrier polypeptide, wherein the nucleic acid comprises a selector codon that is recognized by the O-tRNA, and wherein the nucleic acid is encoded in a cassette on a second plasmid and stably integrated into the cell genome; and, (b) translating the nucleic acid, thereby incorporating the first unnatural amino acid into the carrier polypeptide during translation in the methylotrophic yeast cell; and, reacting the first and second unnatural amino acid residues via in vitro oxime ligation to produce the covalently coupled carrier polypeptide-target polypeptide conjugate.