PMID: 8944787Nov 1, 1996Paper

Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor

European Journal of Biochemistry
J AnagliE Carafoli

Abstract

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.

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Citations

Dec 14, 2004·Chembiochem : a European Journal of Chemical Biology·Norbert SchaschkeChristian P Sommerhoff
Jul 24, 2002·Protein Expression and Purification·Jinyang CongDarrel E Goll
Feb 15, 2002·Biological Chemistry·D Gabrijelcic-GeigerE A Auerswald
Jun 13, 1998·The Journal of Cell Biology·D A PotterI M Herman
May 10, 2011·Biochimica Et Biophysica Acta·Jordan S ChouPeter L Davies
Jun 23, 2010·Meat Science·Elisabeth Huff LonerganSteven M Lonergan
May 28, 1998·Cell Calcium·L SantellaE Carafoli
Oct 6, 2012·The Biochemical Journal·Robert L Campbell, Peter L Davies
Nov 2, 2005·Biochemical and Biophysical Research Communications·Matthew GarciaJames W Geddes
Jul 5, 2003·Physiological Reviews·Darrell E GollJinyang Cong
Jun 8, 2004·The Biochemical Journal·Amaury Fernández-MontalvánWerner Machleidt
Dec 26, 2002·The Journal of Biological Chemistry·Russell BettsJohn Anagli
Mar 20, 1999·Biochimica Et Biophysica Acta·P BessièreF Bancel

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