Purification of N-terminal His-tagged E-Syt1

E-Syt1 SMP-C2ABCDE, E-Syt1 C2ABCDE, or E-Syt1 C2E was expressed in Expi293 cells with an N-terminal His₆-tag, as described previously^{4 (link),13 (link)}. Briefly, cells were harvested by centrifugation at 300 x g for 5 min and lysed by three freeze–thawing cycles in buffer A [25 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1× complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM TCEP] using liquid nitrogen. The lysates were clarified by centrifugation at 17,000 x g for 30 min, and the protein was isolated by a Ni-NTA column (Clontech). Gel filtration (Superdex 200, GE Healthcare) was used to further purify the protein in buffer B (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM TCEP). Pool and concentrate the fractions containing E-Syt1 to ~1 mg/ml.