Modified aminoacyl-tRNA synthetase and use thereof

Patent metadata

Field	Value
Publication number	US-10815489-B2
Application number	US-201615557532-A
Country	US
Kind code	B2
Filing date	Mar 11, 2016
Priority date	Mar 13, 2015
Publication date	Oct 27, 2020
Grant date	Oct 27, 2020

Abstract

The present invention provides modified aminoacyl-tRNA synthetases (ARSs) having increased reactivity with N-methyl amino acids compared to natural aminoacyl-tRNA synthetases. The modified aminoacyl-tRNA synthetases according to the present invention can aminoacylate tRNAs with their corresponding N-methyl-substituted amino acids such as N-methyl-phenylalanine, N-methyl-valine, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan, and N-methyl-leucine more efficiently than natural aminoacyl-tRNA synthetases. The present invention enables a more efficient production of polypeptides containing N-methyl amino acids.

First claim

The invention claimed is:

1. A valyl-tRNA synthetase (ValRS) polypeptide modified to enhance aminoacylation reaction with N-methyl-valine, wherein the polypeptide has aminoacyl-tRNA synthetase (ARS) activity, and wherein the modification comprises at least one amino acid substitution at a position(s) corresponding to asparagine at position 43 and/or threonine at position 45 of ValRS from Escherichia coli.

2. The polypeptide according to claim 1 , wherein the modification further comprises a substitution at a position corresponding to threonine at position 279 of ValRS from Escherichia coli.

3. The polypeptide according to claim 1 , wherein the polypeptide is selected from the group consisting of the following (a) and (b):
(a) a polypeptide comprising amino acids selected from the group consisting of SEQ ID NOs: 3-5, 182, and 183, and
(b) a polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5, 182, and 183.

4. A polynucleotide encoding the polypeptide according to claim 1 .

5. A vector comprising the polynucleotide according to claim 4 .

6. A host cell comprising a polynucleotide encoding a polypeptide according to claim 1 .

7. A method for producing the polypeptide according to claim 1 , comprising the step of culturing a host cell comprising a polynucleotide encoding the polypeptide according to claim 1 .

8. A method for producing a tRNA acylated with N-methyl-valine, comprising the step of contacting N-methyl valine with tRNA in the presence of the polypeptide according to claim 1 .

9. A method for producing a polypeptide comprising N-methyl-valine, comprising the step of performing translation in the presence of the polypeptide according to claim 1 and N-methyl-valine.

10. The method according to claim 9 , wherein the step of performing translation is carried out in a cell-free translation system.

11. The polypeptide according to claim 1 , having (a) glycine or alanine at a position corresponding to asparagine at position 43 and/or (b) serine at a position corresponding to threonine at position 45 of ValRS from Escherichia coli.

12. The polypeptide according to claim 2 , having (a) glycine or alanine at a position corresponding to asparagine at position 43 and/or (b) serine at a position corresponding to threonine at position 45 and/or (c) glycine or alanine at a position corresponding to threonine at position 279 of ValRS from Escherichia coli.

13. A host cell comprising a polynucleotide encoding a polypeptide according to claim 2 .

14. A method for producing a tRNA acylated with N-methyl-valine, comprising the step of contacting N-methyl-valine with tRNA in the presence of the polypeptide according to claim 2 .

15. A method for producing a polypeptide comprising N-methyl-valine, comprising the step of performing translation in the presence of the polypeptide according to claim 2 and the N-methyl-valine.

16. A host cell comprising a polynucleotide encoding a polypeptide according to claim 11 .

17. A method for producing a tRNA acylated with N-methyl-valine, comprising the step of contacting N-methyl-valine with tRNA in the presence of the polypeptide according to claim 11 .

18. A method for producing a polypeptide comprising N-methyl-valine, comprising the step of performing translation in the presence of the polypeptide according to claim 11 and the N-methyl-valine.

19. A host cell comprising a polynucleotide encoding a polypeptide according to claim 12 .

20. A method for producing a tRNA acylated with N-methyl-valine, comprising the step of contacting N-methyl-valine with tRNA in the presence of the polypeptide according to claim 12 .

21. A method for producing a polypeptide comprising N-methyl-valine, comprising the step of performing translation in the presence of the polypeptide according to claim 12 and the N-methyl-valine.

Assignees

• Chugai Pharmaceutical Co Ltd

Inventors

• Ohta Atsushi
• Yamagishi Yusuke
• Matsuo Atsushi

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12P21/00

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

• C12N9/00

  Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00) · CPC title

• C12N5/10

  Cells modified by introduction of foreign genetic material · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title