Purification of AcrB Transporter Protein

The protein was expressed using auto-induction and membrane preparation, styrene maleic acid (SMA) solubilisation and protein purification were performed using the protocols previously reported [36 (link),37 (link)]. Cells were disrupted with a cell disrupter (Constant Systems Ltd.) at 30 kpsi after resuspension in 20 mM Tris, 0.5mM EDTA pH 7.9 (4 °C) in a ratio of 4:1 buffer to pellet. The cell membranes were isolated by differential centrifugation as previously described [38 (link)]. AcrB was extracted in its native trimeric form (~340kDa) using 2.5% (w/v) SMA, incubated for 2 h at room temperature as previously described [36 (link),39 (link)]. The insoluble material was removed by centrifugation (100,000× g, 1 h, 4 °C). The supernatant was mixed with 2 mL HisPur™ Cobalt Resin (Thermo scientific) and incubated in batch overnight at 4 °C with gentle agitation. Following packing, the resin was washed twice with wash buffer (50 mM Tris-HCl, 500 mM NaCl, 10% (w/v) glycerol). After elution (50 mM Tris-HCl, 500 mM NaCl, 10% (w/v) glycerol, 300 mM imidazole), the peak fractions were pooled out and dialysed against a buffer free of imidazole.