Patent ID: 8357512

Claim:
A method for producing, in a host cell, a polypeptide of interest comprising an unnatural amino acid at a specified position, the method comprising: (a) providing: (i) an unnatural amino acid; (ii) a nucleic acid construct comprising a nucleotide sequence encoding an orthogonal tRNA (O-tRNA) and a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein said O-RS preferentially aminoacylates said O-tRNA with said unnatural amino acid; and, (iii) a polynucleotide encoding said polypeptide of interest, said polynucleotide comprising at least one selector codon that is recognized by said O-tRNA, and wherein the position of the selector codon correlates to the specified position of the unnatural amino acid in the polypeptide of interest; (iv) a host cell comprising (i), (ii) and (iii); and wherein said nucleic acid construct of (ii) and (iii) collectively comprises providing at least one of: (I) promoter and terminator nucleotide sequences derived from an Escherichia coli proline tRNA gene, wherein said promoter and terminator sequences are both operatively linked to said nucleotide sequence encoding said O-tRNA, and wherein said nucleotide sequence encoding said O-tRNA is heterologous to said promoter and terminator sequences; or (II) a polycistronic operon comprising a plurality of O-tRNA gene nucleotide sequences, wherein at least one O-tRNA gene is separated from at least one adjacent O-tRNA gene by a heterologous polynucleotide linker derived from a naturally occurring polynucleotide linker from a naturally occurring tRNA operon; and (III) a promoter nucleotide sequence corresponding to a modified E. coli glnS promoter having the nucleotide sequence of SEQ ID NO: 13, wherein said modified E. coli glnS nucleotide sequence is operatively linked to said nucleotide sequence encoding said O-RS; (b) growing said host cell; and (c) incorporating said unnatural amino acid at said specified position in said polypeptide during translation of said polypeptide in said host cell, thereby producing said polypeptide of interest comprising said unnatural amino acid at the specified position.