Patent ID: 8198044

Claim:
A translation system for the expression of a polypeptide of interest comprising at least one unnatural amino acid at a specified position, the system comprising: (a) an unnatural amino acid; (b) a nucleic acid construct, said construct comprising a nucleotide sequence encoding an orthogonal tRNA (O-tRNA) and a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase (O-tRNA), wherein said O-tRNA preferentially aminoacylates said O-tRNA with said unnatural amino acid, wherein said nucleic acid construct comprises at least one of: (i) a promoter and terminator nucleotide sequences from an Escherichia coli proline tRNA gene, wherein said promoter and terminator sequences are both operatively linked to said nucleotide sequence comprising or encoding said O-tRNA, and wherein said O-tRNA is heterologous to said promoter and terminator nucleotide sequences; or a polycistronic operon with a plurality of nucleic acid sequences comprising Methanococcus jannaschii suppressor tyrosyl-tRNA (MjtRNA-Tyr Sup ), wherein at least one MjtRNA-Tyr Sup sequence 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 wherein said polycistronic operon is under the control of a proline promoter and terminator from E. coli; (ii) a nucleotide sequence corresponding to a modified E. coli glnS promoter having a 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; and (c) a polynucleotide encoding said polypeptide of interest, said polynucleotide comprising at least one selector codon that is recognized by said O-tRNA, wherein the position of the selector codon in the polynucleotide controls the specified position of the unnatural amino acid in the polypeptide of interest upon expression of the polynucleotide to produce the polypeptide.