Figure 5. Resurrection of the CspA active site does not restore protease activity in E. coli.

Purification of CspBA variants from the soluble fraction. Cultures expressing the cspBA variants were induced with IPTG overnight at 18°C, and aliquots were removed for analysis of the ‘induced' fraction compared with the ‘uninduced' fraction prior to the addition of IPTG (-IPTG). Cultures were harvested, and cells were lysed using sonication. Following a high-speed centrifugation, the cleared lysate containing soluble proteins was incubated with Ni²⁺-NTA agarose beads. CspBA-His₆ variants were eluted from the beads using imidazole (elution fraction). Samples were resolved by SDS–PAGE and analyzed by Coomassie staining (top) and western blotting (bottom). Non-specific proteins pulled-down with the Ni-NTA beads are marked with asterisks; a truncated CspBA Q757H variant is also marked. An anti-His₆ antibody was used to detect full-length CspBA. An anti-CspB antibody was used to detect CspBA-His₆ variants.