Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

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.