Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Minchen Chien; Thomas K. Anderson; Steffen Jockusch; Chuanjuan Tao; Shiv Kumar; Xiaoxu Li; James J. Russo; Robert Kirchdoerfer; Jingyue Ju

SARS-CoV-2 nsp7 and nsp8: The SARS-CoV-2 nsp7 and nsp8 genes were codon optimized and cloned into pET46 (Novagen) with an N-terminal 6x histidine tag, an enterokinase site, and a TEV protease site. Rosetta2 pLys E. coli cells (Novagen) were used for bacterial expression. After induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), cultures were grown at 16°C for 16 hrs. Cells were harvested by centrifugation and pellets were resuspended in wash buffer (10mM Tris pH 8.0, 300 mM NaCl, 30 mM imidazole, 2 mM DTT). Cells were lysed via microfluidization and lysates were cleared by centrifugation and filtration. Proteins were purified using Ni-NTA agarose beads and eluted with wash buffer containing 300 mM imidazole. Eluted proteins were further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Life Sciences). Purified proteins were concentrated by ultrafiltration prior to flash freezing with liquid nitrogen.