Expression and purification of BamA, OmpX and tOmpA

BamA, OmpX and tOmpA were expressed as inclusion bodies in E. coli BL21(DE3) cells, using a procedure modified from McMorran et al. ⁵⁰. Briefly, inclusion bodies were solubilised in 25 mM Tris–HCl pH 8.0, 6 M guanidine–HCl and were centrifuged (20,000×g, 20 min, 4 °C) to remove remaining insoluble material. The solubilised inclusion bodies were purified by SEC using a Superdex 75 HiLoad 26/60 column (GE Healthcare) for tOmpA and OmpX, and Sephacryl 200 26/60 column for BamA, equilibrated in 25 mM Tris–HCl pH 8.0, 6 M guanidine–HCl. For folding experiments, OmpX and tOmpA were buffer exchanged into Tris-buffered saline (TBS, 20 mM Tris–HCl, 150 mM NaCl) pH 8.0, 8 M urea using Zeba™ Spin Desalting Columns, 7k MWCO, 0.5 mL (Thermo Scientific). BamA was refolded in LDAO detergent prior to reconstitution into proteoliposomes, as described previously⁶² (see below).