Expression and purification of OprF/I and PopB/PcrH from E. coli.

Purification of OprF/I was performed as previously described in patent filings (https://patents.google.com/patent/EP2686339A1/). Briefly, E. coli BL21(DE3) carrying pET24a(+)-OPRF/I was grown in 2 L of fresh LB media with kanamycin and grown at 37°C. When an OD₆₀₀ of 0.8 was reached, the culture was induced with 1 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) and incubated for an additional 3.5 h. E. coli cells were then harvested using centrifugation for 10 min at 7,800 g in 4°C. Cells were resuspended in lysis buffer (buffer A) containing 8 M urea, 20 mM Tris-HCl, 100 mM KCl, 200 mM NaCl, and 10 mM imidazole and then sonicated. Lysates of cells were centrifuged at 10,000 g for 30 min and supernatants were purified through an IMAC column. Column resin was washed with 50 column volumes of buffer A containing 0.1% Triton X-114 followed by 20 column volumes of buffer A. OprF/I was then eluted in buffer A containing 250 mM imidazole. Fractions of elution underwent refolding dialysis with urea, endotoxin removal with polylysine resin, and then final dialysis with reoxidation of the purified material using 1 mM dithiothreitol (DTT). PopB/PcrH was purified as previously described (23).