Patent ID: 8658395

Claim:
A method of reacting a peptide comprising a 5-hydroxy-L-tryptophan (5-HTPP) with a molecule reactive with 5-HTPP, the method comprising: providing a peptide comprising one or more unnatural amino acids wherein at least one of the unnatural amino acids is 5-hydroxy-L-tryptophan (5-HTPP), providing a reactive molecule reactive with the 5-HTPP; and, reacting the reactive molecule covalently with the 5-HTPP, wherein said providing the peptide comprises: i) 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-HTPP when the reference O-tRNA, 5-HTPP, and the O-RS are present in a eukaryotic cell; ii) 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 a 5-HTPP by a reference O-RS of SEQ ID NO: 2 when the reference O-RS, 5-HTPP, and the O-tRNA are present in a eukaryotic cell; iii) introducing the O-RS construct and the O-tRNA construct into the eukaryotic cell; and, iv) preferentially aminoacylating an expressed O-tRNA with the 5-HTPP, 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-HTPP is incorporated into the peptide in the eukaryotic cell.