Polypeptide cleavage method using OmpT protease variant

Patent metadata

Field	Value
Publication number	US-9657323-B2
Application number	US-57382104-A
Country	US
Kind code	B2
Filing date	Sep 29, 2004
Priority date	Sep 30, 2003
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

A polypeptide cleavage method characterized in that arginine or lysine is at the P1 position of a desired cleavage site in a polypeptide, an amino acid other than aspartic acid, glutamic acid or proline is at the P1′ position, a single basic amino acid or two or three consecutive basic amino acids are situated at any site in the amino acid sequence from the P10 position to the P3 position or from the P3′ position to the P5′ position (with the proviso that a single basic amino acid is not situated at the P6 or P4 position), and OmpT protease or its variant enzyme having a substitution at the 97th amino acid from the N-terminus is used to cleave the desired cleavage site in the polypeptide.

First claim

What is claimed is:

1. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the 97th position of the amino acid sequence of SEQ ID NO: 41,
wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine,
wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1′ position, and
wherein the P1 position is arginine or lysine and the P1′ position is:
(1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine;
(2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is methionine; or
(3) alanine, valine, isoleucine, methionine, serine, threonine, cysteine, or asparagine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine.

2. The process of claim 1 , wherein the amino acid sequence from the P10 position to the P3 position comprises only a single basic amino acid or only two or three consecutive basic amino acids.

3. The process of claim 2 , wherein the basic amino acids are arginine and/or lysine.

4. The process of claim 3 , wherein the basic amino acids are arginine.

5. The process of claim 1 ,
wherein the polypeptide is a fusion protein comprising a protecting peptide and a target peptide,
wherein the C-terminus of the protecting peptide is the P1 position and the N-terminus of the target peptide is the P1′ position,
wherein the fusion protein is produced by expressing a gene encoding the fusion protein in a host cell, and
wherein cleavage of the fusion protein liberates the target peptide.

6. The process of claim 5 , wherein a single basic amino acid or two or three consecutive basic amino acids are situated in the amino acid sequence from the P10 position to the P3 position.

7. The process of claim 5 , wherein the E. coli OmpT protease variant is produced by expressing a gene encoding the E. coli OmpT protease variant in said host cell.

8. The process of claim 1 , wherein two or three consecutive basic amino acids are situated between the P10 and P3 positions in the polypeptide.

9. The process of claim 8 , wherein three consecutive basic amino acids are situated between the P5 and P3 positions in the polypeptide.

10. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the position of the amino acid sequence of SEQ ID NO: 41,
wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine,
wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1′ position, and
wherein the P1 position is arginine or lysine and the P1′ position is:
(1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine;
(2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E.coli OmpT protease variant is methionine: or
(3) alanine, valine, isoleucine, methionine, serine, threonine, cysteine, or asparagine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine, and

wherein the amino acid sequence from the P5 to P1 positions in the polypeptide is Arg-Arg-Arg-Ala-Arg (SEQ ID NO: 11).

11. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the 97the position of the amino acid sequence of SEQ ID NO: 41,
wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine,
wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1′ position, and
wherein the P1′ position is:
(1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine;
(2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is methionine; or
(3) alanine, valine, isoleucine, methionine, serine threonine, cysteine, or asparagine, when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine, and

wherein the amino acid sequence from the P7 to P1 positions in the polypeptide is Asp-Ala-Arg-Arg-Arg-Ala-Arg (SEQ ID NO: 12).

12. The process of claim 5 , wherein the target peptide consists of between 22 and 45 amino acid residues.

13. The process of claim 12 , wherein the target peptide is adrenocorticotropic hormone (1-24), motilin, or calcitonin precursor.

Assignees

• Okuno Kazuaki
• Yabuta Masayuki
• Daiichi Sankyo Co Ltd

Inventors

• Okuno Kazuaki
• Yabuta Masayuki

Classifications

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C07K14/695

  Corticotropin [ACTH] · CPC title

• C12P21/06 Primary

  produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) · CPC title

• C07K14/63

  Motilins · CPC title

• C07K14/585

  Calcitonins · CPC title