Recombinant t- and v-SNARE proteins were expressed and purified as we previously described.13 (link),16 (link) The synaptic exocytic t-SNARE complex was composed of untagged rat syntaxin-1 and mouse SNAP-25 with an N-terminal His6 tag. The GLUT4 exocytic t-SNARE complex was composed of untagged rat syntaxin-4 and mouse His6-tagged SNAP-23. Recombinant v-SNARE proteins had no extra residues left after the tags were removed. The v-SNARE mutants were generated by site-directed mutagenesis and purified similarly to WT proteins. SNAREs were stored in a buffer containing 25 mM HEPES (pH 7.4), 400 mM KCl, 1% n-octyl-β-d -glucoside (OG), 10% glycerol, and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP). Soluble factors were stored in the protein binding buffer (25 mM HEPES [pH 7.4], 150 mM KCl, 10% glycerol, and 0.5 mM TCEP).
Recombinant untagged Munc18–1 and Munc18c proteins were produced in E. coli and Sf9 insect cells, respectively, using procedures we previously established.8b (link),11a (link),15a (link),17 (link) To preserve their maximum activities, purified SM proteins were immediately frozen, stored at −70 °C, and used within one month of purification. Full-length (FL) rat synaptotagmin-1 was expressed and purified in the similar way as we described for VAMP2. Human complexin-1 was expressed and purified using the protocol of Munc18–1 preparation.
Recombinant untagged Munc18–1 and Munc18c proteins were produced in E. coli and Sf9 insect cells, respectively, using procedures we previously established.8b (link),11a (link),15a (link),17 (link) To preserve their maximum activities, purified SM proteins were immediately frozen, stored at −70 °C, and used within one month of purification. Full-length (FL) rat synaptotagmin-1 was expressed and purified in the similar way as we described for VAMP2. Human complexin-1 was expressed and purified using the protocol of Munc18–1 preparation.
