Open AccessPurification of Active Calpain by Affinity Chromatography on an Immobilized Peptide Inhibitor
Author(s) -
Anagli John,
Vilei Edy M.,
Molinari Maurizio,
Calderara Silvio,
Carafoli Ernesto
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00948.x
Subject(s) - chemistry , calpain , peptide , chromatography , sepharose , affinity chromatography , calpastatin , elution , egta , amino acid , biochemistry , ion chromatography , enzyme , calcium , organic chemistry
Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide‐affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr‐activated Sepharose® 4B. Crude preparations of calpain, isolated by anion‐exchange chromatography on a DEAE‐Sepharose® column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80‐kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA‐containing buffer. Using the two peptide‐affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30‐kDa heterodimer or in the form of dissociated monomers.