Recombinant Protein Purification and Antibody Generation

The N-001 was prepared as described previously^{24 (link),25 (link)}. In brief, plasmid encoded constructs for C2I and C2II-C1 were expressed in E. coli BL21. E. coli was cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C and were induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). A French pressure cell was used to lyse the cell paste at 690 bar. 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^{45 (link)}. A custom-made polyclonal antibody targeting the N-001 enzymatic component C2I, was prepared in rabbits. The other antibodies used were goat anti-mouse IgG (H/L) polyclonal antibody (BioRad, Cat no. STAR207P) and goat anti-rabbit IgG (H/L): HRP (BioRad, Cat no. STAR124P). Thermo Scientific Pierce TMB substrate was used for ELISAs.