FIGURE 2.

VesB purification by benzamidine-Sepharose affinity chromatography. A, the supernatants of ΔvesABC strains with pMMB67EH (p) or pVesB were incubated for 10 min at 37 °C with different concentrations of the serine protease inhibitor, benzamidine, and the protease activity was measured (experiments were done in triplicates and S.E. bars are shown). B–D, the supernatant from ΔvesABC overexpressing VesB (lane 1) was added to 60% saturation of ammonium sulfate and the precipitated material (Lane 2) and supernatant (Lane 3) were separated by centrifugation. The pellet was resuspended and dialyzed in 50 mm Tris-HCl, pH 8.0, 450 mm NaCl. The sample was used for affinity chromatography using a benzamidine-Sepharose column. Flow-through fraction was discarded (lane 4) and the column was washed with 50 mm Tris-HCl, pH 8.0, 450 mm NaCl (lanes 5–10). VesB was eluted using 100 mm benzamidine (lanes 11–14). The samples were analyzed by SDS-PAGE and GelCode blue staining (B), silver staining (C), or Western blotting with anti-VesB antibodies (D).