Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography

Specific modified substrate-analogous amino acids and peptides have been us ed as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a memb rane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl keton es as affinity ligands have been used to separate subtilisin enzymes and th e cysteine proteases cathepsin B, L, and S. As a new type of ligands, space r-bound peptidyl chloromethyl ketones are presented for a specific and orie nted immobilization of proteinases. Oriented-immobilized cathepsin B was us ed to isolate antibodies against this enzyme. (C) 2001 Elsevier Science BN. All rights reserved.