Purification of Calpain-3 Enzyme

Calpain-3 and calpain-3/titin-I81-I83 complex were purified from lysed E. coli using protocols developed for calpain-2 isolation (38 (link)) with some modifications. Briefly, cells were lysed by sonication in buffer A [50 mM Tris-HCl (pH 7.6), 5.0 mM EDTA, 10 mM 2-mercaptoethanol] containing one tablet of protease inhibitor cocktail (Roche), which can inhibit a broad spectrum of serine and cysteine proteases, and the cell debris were removed through centrifugation at 48,000 x g for 60 min. The supernatant was applied to a DEAE ion-exchange column that had been pre-equilibrated with buffer A. After washing away unbound material, calpain-3 was eluted from the column using a gradient of 0 - 0.75 M NaCl in buffer A. Fractions containing calpain-3 based on SDS-PAGE analysis were combined and MgCl₂ was added with gentle stirring to a final concentration of 23 mM in order to saturate the 5 mM EDTA and avoid stripping Ni²⁺ from the Ni-NTA resin used in the next purification step. The sample was applied to a Ni-NTA column, washed with 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 5 mM imidazole, and eluted wash buffer supplemented with 250mM imidazole.. Elution fractions containing calpain-3 according to SDS-PAGE were dialyzed into a buffer containing 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 2mM EDTA, 10 mM 2-mercaptoethanol, 0.05% Na-azide, and two tablets of protease inhibitor cocktail. Lastly, the calpain-3-enriched sample was applied to a Superdex 200 column (Amersham Biosciences) for a final purification step. Protein purity was assessed using SDS-PAGE visualized by Coomassie Blue. All purification procedures were performed at 4 °C.