Pyrrolysyl-tRNA synthetase

Patent metadata

Field	Value
Publication number	US-12570969-B2
Application number	US-201917271818-A
Country	US
Kind code	B2
Filing date	Aug 30, 2019
Priority date	Aug 31, 2018
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

Abstract

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 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 contacting PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales 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 non-canonical amino acid incorporation system containing highly concentrated PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales.

First claim

The invention claimed is:

1 . 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 reaction solution for a cell-free protein synthesis system, and the reaction solution comprises 25-μm or higher 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, 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.

2 . The production method according to claim 1 , wherein the contact incorporates the non-canonical amino acid into the polypeptide with efficiency at least 1.5 times than efficiency when MmPylRS is used as the PylRS.

3 . The production method according to claim 1 , wherein the reaction solution comprises a polynucleotide encoding a gene having a stop codon at a position different from a naturally occurring position.

4 . The production method according to claim 1 , further comprising a step of mixing a solution containing the wild-type or mutant PylRS of at least 5 mg/mL and the non-canonical amino acid to prepare a mixed solution.

5 . The production method according to claim 1 , wherein the wild-type or mutant PylRS is PylRS from Methanomethylophilus alvus (MaPylRS).

6 . The production method according to claim 1 , wherein the wild-type or mutant PylRS has pyrrolysyl-tRNA synthetase activity, and has (i) the amino acid sequence set forth in SEQ ID NO: 5, or (ii) an amino acid sequence having a mutation at position 96, 120, 121, 122, 125, 126, 128, 129, 164, 166, 168, 170, 203, 204, 205, 206, 207, 221, 223, 227, 228, 233, 235, 239, 241, or 243 of the amino acid sequence set forth in SEQ ID NO: 5.

7 . The production method according to claim 1 , wherein the polypeptide containing a non-canonical amino acid is bonded to a drug.

8 . The production method according to claim 1 , wherein the wild-type or mutant PylRS has an amino acid sequence with at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and has pyrrolysyl-tRNA synthetase activity.

Assignees

• Riken

Inventors

• Sakamoto Kensaku
• Yamaguchi Atsushi
• Kimura Fumie
• Yokoyama Shigeyuki
• Yanagisawa Tatsuo
• Seki Eiko
• Kuratani Mitsuo

Classifications

• C12Y601/01026

  Pyrrolysine-tRNAPyl ligase (6.1.1.26) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/93 Primary

  Ligases (6) · CPC title