Polypeptide cleavage method using OmpT protease variant
AssigneeDaiichi Sankyo Company, Limited
InventorsKazuaki Okuno, Masayuki Yabuta
Life of this patent
Estimating the term…
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.
Claims13
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.
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).
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).
Dependencies are inferred from the claim text.