Purification of SNARE Complex Proteins

syt1, cpx2, EIIA^Glc, MSP1D1, and SNAREs were expressed in BL21 STAR^TM (DE3) and purified using GSTrap and Ni²⁺-NTA columns^{20 (link),25 (link),44 (link)–47 (link)}. To produce spMSP1D1,spNW15, spNW25, spNW30, spNW50, spNW80, and spNW100, plasmids were transformed into BL21 STAR^TM (DE3) cells that were grown in LB supplemented with Km (50 mg/ml) to OD₆₀₀ ~ 0.7. Protein expression was induced with 0.2 mM IPTG at 16 °C overnight. Bacteria were harvested by centrifugation at 3450 × g for 20 min, resuspended in buffer A (50 mM Tris-HCl (pH 8),100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), and lysed on ice using a Branson cell disrupter (60% duty cycle, 45 secs). Cell lysates were clarified by centrifugation at 12,000 × g for 45 mins. The supernatants were loaded onto a 1 ml Ni²⁺-NTA column (GE Healthcare), followed by extensive wash (20 column volume) using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), desalted in buffer A using PD MiDiTrap G-25 (GE Healthcare), and stored at −80 °C.