Protease activity of rHtrA and its analogs. (A) Wild-type rHtrA (0.5 μg) was mixed with β-casein (5 μg) and incubated at 37°C. Samples were taken at various time points and analyzed by SDS-PAGE. Lane 1, molecular weight standards; lane 2, t = 0 min; lane 3, t = 10 min; lane 4, t = 20 min; lane 5, t = 30 min; lane 6, t = 1 h; lane 7, t = 2 h; lane 8, t = 4 h; lane 9, t = 16 h; lane 10, no HtrA added at t = 16 h. (B) Wild-type rHtrA or its analogs (0.5 μg) were mixed with β-casein (5 μg) and incubated at 37°C for 16 h. Lane 1, molecular weight standards; lane 2, casein alone; lane 3, rHtrA; lane 4, S197A; lane 5, S197C; lane 6, S197T; lane 7, H91A; lane 8, H91R; lane 9, D121A; lane 10, D121E. For both panels, the numbers at the left represent molecular weights in thousands.