Figure 3.

Expression, purification, and the effect of calcium on autoprocessing of mirolysin without C-terminal extension (CTE). A) Catalytic domain preceded by the prodomain of mirolysin and its active site mutein (Mircat and Mircat^E225A, respectively) were obtained as recombinant proteins and purified by affinity chromatography on glutathione-Sepharose with simultaneous removal of the GST tag by PreScission cleavage followed by size exclusion chromatography (SEC) and ion exchange chromatography (Mircat^E225A only). Lanes 1 and 2, E. coli extracts before and 8 h after induction of protein expression with IPTG, respectively; lanes 3 and 4, proteins after purification by affinity chromatography and SEC followed by ion exchange chromatography for Mircat^E225A only, respectively. B-E) Mircat was incubated at 0.5 mg/ml in 50 mM Tris, 0.02% NaN₃ pH 8.0, alone (B) or supplemented with 2.5 mM CaCl₂ (D). At specific time points, aliquots were taken and analyzed by SDS-PAGE (B, D) followed by zymography (C, for incubation in calcium-free buffer only) and measurement of residual activity against Azocoll as a substrate (E). N-terminal sequences determined by Edman degradation are shown on the right.