Purification of SNARE Proteins

Neuronal SNAREs from Rattus norvegicus: SNAP-25 (amino acids 1–206), Syb2 (amino acids 1–116) and Stx1 (amino acids 1–288) were expressed in Escherichia coli CodonPlus-RIL (DE3) and purified by a glutathione S-transferase (GST) tag system. In brief, cell pellets were resuspended in PBS (pH 7.4) supplemented with 2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM dithiothreitol (DTT). After sonication, the supernatant was mixed with GST-agarose beads at 4°C for 3 h. Excess PBS was used for washing, and each protein of interest was eluted in thrombin cleavage buffer (TCB, 50 mM Tris-HCl and 150 mM NaCl, pH 8.0). For transmembrane proteins, 0.2% Triton X-100 and 0.05% Tween 20 were added to PBS for the lysis and washing steps, and subsequently 1% n-octyl-beta-D-glucopyranoside (OG) was added to TCB instead of Triton X-100 at the elution step. All purified proteins were analyzed by SDS-PAGE and the Bradford assay.