Patent ID: 8647840

Claim:
A method for producing, in a methylotrophic yeast cell, a polypeptide of interest comprising an unnatural amino acid at a selected position, the method comprising: a) providing a methylotrophic yeast cell comprising: i) an unnatural amino acid; ii) an orthogonal aminoacyl-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 FLD1 promoter; and, 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 unnatural amino acid in the methylotrophic yeast cell; and, iv) a nucleic acid of interest encoding a polypeptide of interest, wherein the nucleic acid of interest comprises at least one selector codon that is recognized by the O-tRNA, and wherein the nucleic acid of interest is encoded in a cassette on a second plasmid and stably integrated into the cell genome; and, b) incorporating the unnatural amino acid at a selected position in the nucleic acid of interest during translation of the polypeptide of interest in response to a selector codon, thereby producing the polypeptide of interest comprising the unnatural amino acid at the selected position.