Fig. 2
Expression and purification of the VacA recombinant protein in the pET-41b-vacA34–854- E. coli BL21(DE3) system. A and B The expression of the VacA recombinant protein in E. coli BL21(DE3) cells as detected by SDS-PAGE and western blotting, respectively. NC: no induced cell lysis; Lane 1: 15 °C in the induction of cell lysis of 16 h; and Lane 2: 15 °C in the induction of 16 h of cell lysis supernatant; Lane 3: 15 °C in precipitation of cell lysis induced 16 h. C VacA recombinant protein in the inclusion bodies purified with Ni column (IDA) as detected by SDS-PAGE analysis. Lane 1: Loading; Lane 2: Flow through; Lane 3: Elution with 20 mM imidazole; Lane 4: Elution with 500 mM imidazole; Lane 5: Ni resin after elution. Lane M: Protein marker. D and E VacA recombinant protein in the supernatant purified with Ni column (NTA) as detected by SDS-PAGE and western blotting, respectively. The column equilibration buffer consists of 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, and wash buffer consists of 50 mM Tris-HCl, 1% Triton-X114, 8 M urea, 150 mM Nacl, PH 8.0). The target protein was eluted using a stepwise gradient of imidazole (i.e. 20, 50, 100 and 500 mM) and the results are shown as below: Lane 1: Pellet of cell lysate after centrifugation; Lane 2: Supernatant of cell lysate after centrifugation; Lane 3: Flow through; Lane 3–5: Elution with 20 mM imidazole; Lane 6–7: Elution with 50 mM imidazole; Lane 8–10: Elution with 100 mM imidazole; Lane 11–13: Elution with 500 mM imidazole; Lane 14: Ni resin after elution. Lane M: Protein marker. PC:positive control