Sm0431

After amplification using primers PmrAF and PmrAR, PCR products were inserted into pET-28a(+) digested with NdeI and HindIII to add a carboxyl terminal 6-his-tag. Plasmids were transformed into the expression strain E. coli BL21 (DE3) and cultured in LB medium (5 ml) at 37°C overnight. Cells were diluted 1:100 into 1 L LB broth containing ampicillin and cultured. When OD₆₀₀ was between 0.4 and 0.5, recombinant PmrA was activated with 0.1 mM IPTG at 16°C for 20 h.
Mixtures were centrifuged at 7,000 × g for 20 min at 4°C and resuspended in lysis solution, followed by harvest of 6 L of cells and disruption by a One Shot Cell Disrupter (Constant Systems, Daventry, UK). To isolate insoluble substances, mixtures were passed through 0.45-μm syringe-end filters after centrifuging at 12,000 × g for 20 min at 4°C before loading on a His-tag column. Supernatants were transferred to a Ni-NTA affinity chromatography column (GE Healthcare, Pittsburgh, PA, USA). After balancing the column with lysis buffer, recombinant protein was added and eluted using washing buffer with an imidazole concentration gradient (0, 30, 60, 100, 200, and 500 mM). Washing buffer (30 ml) containing target recombinant proteins was dialyzed in 20 mM Tris-HCl, pH 8.0. Proteins were subjected to SDS-PAGE. Unstained and prestained protein markers (Fermentas, SM0431 and SM0671 Waltham, USA) were applied as references.