Method for the selective cleavage of fusion proteins with lysostaphin

• US 5,270,176 A
• Filed: 06/29/1992
• Issued: 12/14/1993
• Est. Priority Date: 11/20/1987
• Status: Expired due to Term

• Current Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Original Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Inventors: Seipke, Gerhard, Dorschug, Michael
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): Ulm, John D.
• Application Number:
  US07/905,482
• Time in Patent Office: 533 Days
• Field of Search: 435/69.7, 435/172.3, 530/412, 935/47
• US Class Current: 435/69.7
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

The invention relates to a method for the preparation of polypeptides or proteins by enzymatic cleavage of the oligo- or polyglycine sequence of a fusion protein using an endoprotease.

Claims

1. A method for the preparation of proinsulin by enzymatic cleavage of a fusion protein, wherein

(a) a fusion protein is formed by linking proinsulin to a peptide as follows:

space="preserve" listing-type="equation">(Y.sub.1 . . . Y.sub.m) - (Gly).sub.p+q - (X)

where (Y₁ . . . Y_m) - (Gly)_p represents the sequence which is to be eliminated and (Gly)_q -(X) represents proinsulin;

X and Y denote, independently of one another, natural amino acids;

m is greater than 1;

p is greater than 0;

p+q is between 2 and 100, and if Y_m is Gly, p+q can be less than 2, and p can be 0; and

(b) cleaving the fusion protein with lysostaphin.

2. The method as claimed in claim 1, wherein p+q is between 2 and 30.

3. The method as claimed in claim 2, wherein p+q is between 2 and 18.

4. The method as claimed in claim 1, wherein betagalactosidase is employed as the sequence to be eliminated.

5. The method as claimed in claim 4, wherein a segment of beta-galactosidase is linked to proinsulin by a (Gly)₁₈ peptide.