Patent ID: 8173364

Claim:
A cell comprising an orthogonal tRNA synthetase (ORS) and an orthogonal tRNA (OtRNA), as a pair wherein: (a) said ORS preferentially aminoacylates said OtRNA, wherein preferential aminoacylation is defined in that said ORS aminoacylates endogenous tRNAs of the cell with a reduced efficiency as compared to endogenous tRNA synthetase aminoacylation of said endogenous tRNAs of the cell and wherein said ORS is derived from a TyrRS, a LeuRS, or a ThrRS and wherein said OtRNA is derived from a tRNATyr, a tRNALeu, or a tRNAThr, and wherein the ORS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-64 and conservative variants thereof; (b) said ORS preferentially aminoacylates said OtRNA with an unnatural amino acid, as compared to any natural amino acid, and wherein said unnatural amino acid comprises a tyrosine analogue or derivative, a phenylalanine analogue or derivative, a leucine analogue or derivative, or a threonine analogue or derivative; (c) said OtRNA is aminoacylated by the endogenous tRNA synthetases of the cell with reduced efficiency as compared to aminoacylation of the endogenous tRNAs by said endogenous tRNA synthetases; (d) said OtRNA recognizes a selector codon of an mRNA in said cell; and wherein k cat /K m for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than k cat /K m for aminoacylation of the OtRNA by the ORS with the natural amino acid, or wherein said unnatural amino acid is incorporated in to a growing polypeptide in the cell with a fidelity of greater than 75% in response to the selector codon; and, (e) said cells are cultured in the absence of said unnatural amino acid.