Fig. 4

SDS-PAGE analysis and BApNA activity assays of samples collected from small-scale growth experiments of SHuffle® E. coli cells (NEB) grown in TB media at 15 °C (induced with 0.1 mM IPTG). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). a The gel represents the soluble expression of AaET-NL zymogen (MW ~ 27.0 kDa, yellow arrow), auto-activating to the active mature form (MW ~ 22.4 kDa, purple arrow). The presence of active AaET at 5 h post-induction correlates with an increase in BApNA activity, with maximal activity observed at the 24 h time-point (plot on the right). b The gel represents the soluble expression of AaSPVI-NL zymogen (MW ~ 28.7 kDa, yellow arrow), auto-activating to the active mature form (MW ~ 24.1 kDa, purple arrow). The presence of active AaSPVI at 16 h post-induction correlates with an increase in BApNA activity, with maximal activity observed at the 67 h time-point (plot on the right). c The gel represents the soluble expression of AaSPVII-NL zymogen (MW ~ 28.7 kDa, yellow arrow), auto-activating to the active mature form (MW ~ 24.2 kDa, purple arrow). The presence of active AaSPVII at 15 h post-induction correlates with an increase in BApNA activity, with maximal activity observed at the 18 h time-point (plot on the right). Unlike AaET and AaSPVI, AaSPVII expression results in a species that lies between the zymogen and active forms starting at 8 h post-induction and disappearing at 15 h. This species is an inactive form of AaSPVII since no detectable BApNA activity observed