Figure 7

(A) & (B) Limited proteolysis of Rv1694 with Proteinase K. Rv1694 was partially digested with Proteinase K for indicated times, electrophoresed on 12% SDS-PAGE, stained with coomassie brilliant blue R-250. The lanes were marked to indicate M for protein molecular weight markers, numbers represent the time points: 0 for Rv1694 protein only and lanes marked with 0.5, 1, 2, 5, 10, 20 and 30 respectively represent Proteinase K treatment times in minutes. The arrow on the right indicates the stable domains of Rv1694. The panel (B) was obtained by probing with anti-6-histidine monoclonal antibody. (C) Limited proteolysis of Rv1694 bound to rRBC membranes: Purified Rv1694 protein and rRBC were incubated for 30 min and subjected to digestion with Proteinase K for indicated periods of time. At the end of the incubation, the rRBC were mixed with 2 mM PMSF, washed twice with hypotonic buffer, solublized the RBC membranes and were electrophoresed on 12% SDS-PAGE, probed with anti-6-histidine-monoclonal antibody. The lanes marked with Rv1694 and RBC respectively represent Rv1694 protein only and rRBC membrane only. Lanes indicated with 0.5, 1, 2, 5, 10, 20 and 30 respectively represents the Proteinase K treatment times. Results shown are one of the two independent experiments.