Bacterial Protein Purification and Activation

C2C was prepared as described previously^{19 (link)}. Briefly, plasmid encoded expression constructs for C2I and C2II-C1 were expressed in E. coli BL21. Cell lines were grown in LB medium, with ampicillin (100 µg/mL) at 37 °C, and induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM IPTG. A french pressure cell was used to lyse cell paste with 10,000 psi pressure. Glutathione resin (Genscript) was used for affinity purification. GST fusion tags were removed using thrombin (Thermo Fisher). C2II-C1 was further activated using trypsin by incubation at 37 °C for 30 min at a 1:5 enzyme to substrate ratio as previously described^{63 (link)}.