PYRROLYSYL-TRNA SYNTHETASE

Obtained is: a method for efficiently producing a polypeptide containing a non-canonical amino acid; a method for incorporating a non-canonical amino acid into a polypeptide; a method for producing tRNA bound to a non-canonical amino acid; a a non-canonical amino acid incorporation system; a material for use in these methods; or the like. Used is a method for producing a polypeptide containing a non-canonical amino acid, comprising: a step of bringing PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales into contact with the non-canonical amino acid; and an incorporation step selected from step (a) of incorporating the non-canonical amino acid into the polypeptide with higher efficiency than in a case of using a cell-free protein synthesis system with PylRS of Methanosarcina mazei (MmPylRS) or step (b) of incorporating the non-canonical amino acid into the polypeptide with higher efficiency than in a case of using an Escherichia coli protein synthesis system with a vector carrying a gene for the PylRS under regulation by a glmS promoter or a case of using an Escherichia coli protein synthesis system with a vector carrying a gene for the PylRS under regulation by a glnS promoter. Alternatively, used is a a non-canonical amino acid incorporation system containing highly concentrated PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales.