Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

Dene Littler; Benjamin Gully; Rhys N Colson; Jamie Rossjohn

Synthetic cDNA for the Nsp9COV19 protein was cloned into pET28-LIC expression vector bearing an N-terminal His-tag with a Rhinovirus 3C protease cleavage site (MAHHHHHHSAALEVLFQGPG). The plasmid was transformed into E. coli BL21(DE3) cells which were grown in Luria Broth at 37°C until reaching an Absorbance at 600nm of ~1.0 before being induced with 0.5mM Isopropyl β-d-1-thiogalactopyranoside for 4 hours. Cells were harvested in 20mM HEPES pH 7.0, 150mM NaCl, 20mM Imidazole, 2mM MgCl2 and 0.5mM TCEP and frozen until required. Lysis was achieved by sonicating the cells in the presence of 1mg of Lysozyme and 1mg of DNAase on ice. The lysate was then cleared by centrifugation at 10,000xg for 20 minutes and loaded onto a nickel affinity column. Bound protein was washed extensively with 20 column volumes of 20mM HEPES pH 7.0, 150mM NaCl, 0.5mM TCEP before being eluted in the same buffer with the addition of 400mM Imidazole. For His-tag removal samples were incubated with precision 3c protease overnight at 4°C. All samples were subjected to gel filtration (S75 16/60; GE Healthcare) in 20mM HEPES pH 7.0, 150mM NaCl before being concentrated to 50mg/mL for crystallization trials.