Purification of CCHFV nucleocapsid protein

The protein purification was carried out as previously reported [20 (link), 22 (link), 23 (link)]. Briefly, Escherichia coli Rosetta (DE3) cells (Stratagene), transformed with plasmids expressing either wild type CCHFV N protein or stalk domain were grown in 250 ml cultures at 37°C until the OD at 600 nm reached 0.5. The protein expression was induced by the addition of 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) to the bacterial culture, followed by further incubation at 18°C for additional 20 hours. Cell pallets were re-suspended in 45 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM dithiothreitol [DTT], 0.5% Triton-X100, 5mM CHAPS, 0.1 mM phenyl methyl sulfonyl fluoride [PMSF]), followed by sonication on ice and clearance of the lysate by centrifugation at 4500xg for 20 minutes. The protein purification was carried out on AKTA pure protein purification system (GE Healthcare). The cleared cell lysates were loaded onto HisTrap NiNTA column having 5ml bed volume (Sigma), pre-equilibrated with the lysis buffer. The column was then washed with 50 ml of lysis buffer, followed by additional washing with 100 ml of wash buffer (50mM Tris, 500 mM NaCl, 0.05% Triton X-100 and 20 mM Imidazole). The bound protein was finally eluted by Imidazole gradient from 0 mM to 250 mM in the elution buffer (50mM Tris, 500 mM NaCl, 0.05% Triton X-100).