Purification of carboxypeptidase G‐1 by immunoadsorption

Richard Cornell; Stanley E. Charm

doi:10.1002/bit.260180813

$INTRODUCTION Carboxypeptidase G-1 (CPG-l), an enzyme under study as a possible chemotherapy agent, is produced on pilot plant scale at the New England Enzyme Center. This enzyme cleaves glutamic acid carboxyl from folates and methotrexate. It is a constitutive enzyme from a microorganism isolated a t the Enzyme Center but not yet classified and is identical to the enzyme reported by McCullough e t al.1 Purification by immunosorption was studied as a possible large scale isolation method. PROCEDURE Prepardlion of Immunosorbent Antiserum to carboxypeptidase G-1 was produced by inoculating New Zealand rabbits with 0.5 ml of enzyme with a specific activity of between 375 to 520 units/mg protein in complete Freund's adjuvant. Antiserum to enzyme contaminants was also produced by inoculating 0.7 mg of contaminant protein into a rabbit. The rabbit serum was drawn weekly and fractionated to immunoglobulin. The various antisera were attached to newly activated Sepharose 2B, e.g., see ref. 2. The immunosorbent contained about 20 mg protein/g of filtered dry Sepharose. Immunosorption purification of CPG-1 About three parts of crude enzyme solution and one part of immunosorbent were mixed gently for 2 hr to permit the enzyme to complex with the immunosorbent. The suspension was$

Purification of carboxypeptidase G‐1 by immunoadsorption

Cornell, Richard; Charm, Stanley E.

Follow Biotechnology and Bioengineering , Volume 18 (8) Wiley – Aug 1, 1976