Recombinant SARS-CoV-2 Protein Purification

The coding sequences of SARS-CoV nsp10, 16; SARS-CoV-2 nsp10, 14, 16; and mutants were cloned into a pET32a vector with the His tag. E. coli BL21 (DE3) cells were transformed with the respective plasmid and the recombinant protein was induced with 0.4 mM isopropyl β-d-thiogalactopyranoside (IPTG) at 16 °C for 12–16 h. The cells were harvested by centrifugation, and the pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10% glycerol, and 5 mM MgCl₂). The cells were then disrupted by a high-pressure cracker (UH-24, Union-biotech, Shanghai, China), and cell debris was removed by centrifugation. pET32a-His6-nsp10, 14, and 16 were purified with nickel-nitrilotriacetic acid (Ni-NTA, Shanghai, China) resin (GenScript, Piscataway, NJ, USA) as described previously [45 (link),46 (link)].