Genes encoding OmpA, OmpT, and OmpA171 without the signal peptide were amplified from the genomic DNA of E. coli K-12 and inserted into plasmid pET22b, which introduced a six-histidine tag at the C-terminal of protein. Primers used in the construction of plasmids are: 5′-GCT CAT ATG GCT CCG AAA GAT AAC ACC TGG TAC-3′ (OmpA forward), 5′-GCC TCG AGA GCC TGC GGC TGA GTT ACA ACG TC-3′ (OmpA reverse), 5′-GAC ATA TGT CTA CCG AGA CTT TAT CGT TTA CTC-3′ (OmpT forward), 5′-GAC TCG AGA AAT GTG TAC TTA AGA CCA GCA GTA G-3′ (OmpT reverse), 5′-GAC ATA TGG CTC CGA AAG ATA ACA CCT GGT AC-3′ (OmpA171 forward), and 5′-AAC TCG AGA CCG AAA CGG TAG GAA ACA CC-3′ (OmpA171 reverse). Restriction enzyme digestion sites are underlined. Plasmid containing OMP gene was then transformed into E. coli strain ER2566 for protein expression. Cells were cultured at 37°C in Lysogeny Broth media containing 100 μg mL−1 ampicillin to an OD600nm of approximately 0.6. The expression of protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM. After 3 h, cells were harvested by centrifugation at 7000g for 10 min at 4°C. Cell pellets were stored at −20°C or used immediately for purification.
The purification procedures for OmpA and OmpT were the same. Briefly, cell pellets were resuspended in a lysis buffer (30 mM Tris base, 0.5M NaCl, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, pH 8.0) and then sonicated on ice for 15 min with 10-s on/off intervals to lyse cells. After centrifugation at 10,000g for 30 min at 4°C, the supernatant containing soluble proteins was discarded, whereas the pellet was dissolved in the sonication buffer (30 mM Tris base, 0.5M NaCl, 6M guanidine, pH 8.0) and sonicated again with the same setting. After sonication, the mixture was centrifuged at 25,000g for 20 min at 4°C. The supernatant containing solubilized OMPs was loaded to a Ni-nitrilotriacetic acid column, washed using a buffer containing imidazole (30 mM Tris base, 0.5M NaCl, 8M urea, 20mM imidazole, pH 8.0), and eluted using an elution buffer (20mM sodium acetate, 0.5M NaCl, 8M urea, pH 4.0). The pH of protein solution was then adjusted to 8.0 immediately after elution. OmpA171 was purified as described with slight modifications.10 Briefly, cell pellets were resuspended in the lysis buffer, sonicated on ice, and then centrifuged to obtain the inclusion body similarly as described for the purification of OmpA and OmpT. The inclusion body was washed twice using a wash buffer (20 mM sodium acetate, 0.5M NaCl, 2M urea, 1% Triton, pH 8.0) followed by centrifugation (2900g, 30 min, 4°C). Before refolding experiment, OmpA171 pellet was dissolved in a buffer containing 8M urea (2 mM EDTA, and 10 mM borate, pH 10.0). Purified protein was analyzed using SDS–PAGE and visualized after stained using Coomassie blue. Concentrations of all three proteins were determined using Pierce BCA Protein Assay Kit (Thermal Scientific, TX).