Patent ID: 7741071
Filing Date: 2010-06-22
Classification: C07K,C12N,C12P

Abstract:
1. A method of incorporating a 5-substituted tryptophan (HTTP) or a 5-substituted tryptophan unnatural amino acid into a peptide, the method comprising: preparing a construct comprising a nucleic acid sequence encoding an orthogonal tRNA synthetase (O-RS) and comprising at least 90% identity to the sequence of SEQ ID NO: 2, the O-RS comprising a proline residue at a position corresponding to position 144 of SEQ ID NO: 2, wherein the O-RS aminoacylates a reference orthogonal tRNA (O-tRNA) of SEQ ID NO: 3 with a 5-HTTP or a 5-substituted tryptophan analog when the reference O-tRNA, 5-HTTP or 5-substituted tryptophan analog, and the O-RS are present in a eukaryotic cell; preparing a construct comprising a nucleic acid sequence encoding an O-tRNA comprising: at least 90% identity to SEQ ID NO: 3, wherein the O-tRNA is aminoacylated with the 5-HTTP or 5-substituted tryptophan analog by a reference O-RS of SEQ ID NO: 2 when the reference RS, 5-HTTP or 5-substituted tryptophan analog, and the O-tRNA are present in eukaryotic cell; introducing the O-RS construct and the O-tRNA construct into the eukaryotic cell; and, preferentially aminoacylating an expressed O-tRNA with the unnatural amino acid, wherein said aminoacylation is catalyzed by an expressed O-RS, wherein the O-tRNA recognizes a selector codon in a nucleic acid sequence encoding the peptide, whereby the 5-HTTP or 5-substituted tryptophan unnatural amino acid is incorporated into the peptide in the eukaryotic cell.