Recombinant BAX Protein Purification

Recombinant, full-length BAX was expressed in BL21 (DE3) E. coli using the pTYB1 vector^{1 (link),6 (link)}. Cell pellets were resuspended in 20 mM Tris, 250 mM NaCl, pH 7.2 and lysed by two passes through a microfluidizer (Microfluidics) chilled to 4°C. The lysate was clarified by centrifugation at 20,000 rpm. BAX was purified by batch affinity binding at 4°C using chitin resin (New England Biolabs), followed by loading onto gravity flow columns for washing and elution. The intein-chitin binding domain tag was cleaved by 36 hour incubation in 50 mM dithiothreitol at 4°C. Pure protein was isolated by size exclusion chromatography (Superdex 75 10/300; 20 mM HEPES, 150 mM KCl, pH 7.2 or 20 mM potassium phosphate, pH 6.2) using an FPLC system (GE Healthcare Life Sciences).