Pyrrolysyl-trna synthetase

A method for producing a polypeptide containing a non-canonical amino acid, comprising:

• a step of contacting a pyrrolysyl-tRNA synthetase (PylRS) with the non-canonical amino acid;
• wherein:
• the contact is performed in a non-canonical amino acid incorporation system containing the PylRS, the non-canonical amino acid incorporation system comprises a cell for a living-cell protein synthesis system, the cell comprises a polynucleotide encoding the PylRS and a high-expression promoter, the high-expression promoter is linked to the polynucleotide encoding the PylRS so that expression of the PylRS can be regulated, and the non-canonical amino acid incorporation system highly expresses the PylRS,
• the PylRS is a wild-type or mutant PylRS, and the wild-type or mutant PylRS has an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO:
• 5 and has pyrrolysyl-tRNA synthetase activity,
• the high-expression promoter is T3, T5, T7, SP6, tac, trc, lac, lacUV5, rhaBAD, SV40, CMV, CAG, SV40, EF-1α, TEF1, PGK1, HXT7, TPI1, TDH3, PYK1, ADH1, GAL1, GAL10, polyhedrin, p10, metallothionein, or Actin 5C, and
• the non-canonical amino acid is a lysine derivative, tyrosine derivative, phenylalanine derivative, tryptophan derivative, arginine derivative, methionine derivative, leucine derivative, histidine derivative, proline derivative, cysteine derivative, threonine derivative, serine derivative, alanine derivative, isoleucine derivative, valine derivative, glutamine derivative, glutamic acid derivative, asparagine derivative, aspartic acid derivative, glycine derivative, selenocysteine derivative, pyrrolysine derivative, kynurenine derivative, ornithine derivative, citrulline derivative, canavanine derivative, or diaminopimelic acid, or an α-hydroxy acid derivative thereof.

A method for incorporating a non-canonical amino acid into a polypeptide, including the step of expressing, at a high level in a living cell, PylRS, and the step of contacting the PylRS with the non-canonical amino acid,

• wherein the PylRS is a wild-type or mutant PylRS, and the wild-type or mutant PylRS has an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO:
• 5 and has pyrrolysyl-tRNA synthetase activity,
• the non-canonical amino acid is a lysine derivative, tyrosine derivative, phenylalanine derivative, tryptophan derivative, arginine derivative, methionine derivative, leucine derivative, histidine derivative, proline derivative, cysteine derivative, threonine derivative, serine derivative, alanine derivative, isoleucine derivative, valine derivative, glutamine derivative, glutamic acid derivative, asparagine derivative, aspartic acid derivative, glycine derivative, selenocysteine derivative, pyrrolysine derivative, kynurenine derivative, ornithine derivative, citrulline derivative, canavanine derivative, or diaminopimelic acid, or an α-hydroxy acid derivative thereof
• the cell comprises a polynucleotide encoding the PylRS and a high-expression promoter,
• the high-expression promoter is controllably linked to a region of the polynucleotide encoding the PylRS so that expression of the PylRS can be regulated, and
• the high-expression promoter is T3, T5, T7, SP6, tac, trc, lac, lacUV5, rhaBAD, SV40, CMV, CAG, SV40, EF-1α, TEF1, PGK1, HXT7, TPI1, TDH3, PYK1, ADH1, GAL1, GAL10, polyhedrin, p10, metallothionein, or Actin 5C.