Patent ID: 7718410

Claim:
A method of producing a protein comprising an unnatural alkynyl amino acid in a eubacterial cell, where the alkynyl amino acid is at a specified position, the method comprising: (a) providing a eubacterial cell comprising: (i) an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein said O-RS is at least 90% identical to a Methanococcus jannaschii tyrosyl-tRNA synthetase of SEQ ID NO: 2, wherein said O-RS has an amino acid sequence comprising the following: (a) alanine at a position corresponding to position 32 of SEQ ID NO: 2; (b) glutamine at a position corresponding to position 107 of SEQ ID NO: 2; (c) alanine at a position corresponding to position 158 of SEQ ID NO: 2; and (d) alanine or proline at a position corresponding to position 162 of SEQ ID NO: 2; (ii) an orthogonal tRNA (O-tRNA), wherein said O-RS preferentially aminoacylates said O-tRNA with said alkynyl amino acid; (iii) a nucleic acid encoding said protein, wherein the nucleic acid comprises at least one selector codon that is recognized by the O-tRNA; and, (iv) said alkynyl amino acid, which alkynyl amino acid is a para-substituted tyrosine or a para-substituted phenylalanine; and, (b) growing said cell; (c) incorporating said para-substituted tyrosine or para-substituted phenylalanine at said specified position in the protein encoded by the nucleic acid during translation of the protein, wherein the specified position in the protein corresponds to the position of the selector codon in said nucleic acid, thereby producing said protein comprising said alkynyl amino acid at the specified position.