Patent ID: 8071344
Filing Date: 2011-12-06
Classification: C12N,C12P

Abstract:
1. A method of producing a protein selectively labeled at a specified position, the method comprising: growing a cell, in an appropriate medium, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a protein; and, providing a keto derivative of tyrosine or a keto derivative of phenylalanine, wherein the keto derivative is other than a meta-acetyl phenylalanine; wherein the cell further comprises: a first orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon; and, an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates the first O-tRNA with an efficiency of at least 50% compared to the aminoacylation efficiency of a synthetase polypeptide comprising the amino acid sequence of SEQ ID NO: 18 aminoacylates an O-tRNA of SEQ ID NO: 21 with p-acetyl-L-phenylalanine; incorporating the keto derivative into the specified position in the protein during translation of the nucleic acid with the at least one selector codon, thereby producing the protein comprising the keto derivative; and, selectively labeling the protein at the keto derivative; wherein the O-RS comprises the amino acid sequence which comprises any one of SEQ ID NO: 18-20, or a conservative variation thereof comprising at least 95% identity to any one of SEQ ID NO: 18-20; wherein the ORS further comprises an Ala or Leu at a position corresponding to position 32; comprises Gly at a position corresponding to position 158; a Gly, Cys or Thr at a position corresponding to position 159; and comprises an Arg, or Leu at a position corresponding to position 162.