Enzymatic method for modification of recombinant polypeptides

US6403361B1 · kind B1 · utility

Assignee

• Restoragen, Inc. · US

Inventors

• Fred W. Wagner · Walton, US
• Jay Stout · Lincoln, US
• Dennis Henriksen · Lincoln, US
• Bruce E. Partridge · Lincoln, US
• Shane Manning · Lincoln, US

Key dates

• Filing date: Feb 17, 2000
• Grant date: Jun 11, 2002
• Expiry date: Feb 17, 2020

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C07K2319/00
• WIPO field: Pharmaceuticals
• WIPO sector: Chemistry

Abstract

The method of the invention provides for the formation of a recombinant polypeptide which has been modified at the C-terminal end through the use of a transpeptidation process. The method is suitable for modifying recombinant polypeptides of any source including those which may be commercially available, those derived from recombinant single copy or multicopy polypeptide constructs, or those derived from single or multicopy recombinant fusion protein constructs. The transpeptidation reaction involves contacting an endopeptidase enzyme with a recombinant polypeptide to substitute an addition unit, of one or more amino acids, for a leaving unit, linked to a core polypeptide through a cleavage site recognized by the endopeptidase enzyme. Recombinant polypeptides derived from multicopy polypeptide constructs may be cleaved from the multicopy polypeptide at the N-terminal and C-terminal ends and simultaneously under go substitution of the leaving unit by the desired addition unit. The invention utilizes known and newly discovered cleavage recognition sites to effectuate the desired modification products.