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 nsp12: The SARS-CoV-2 sp12 gene was codon optimized and cloned into pFastBac with Cterminal additions of a TEV site and strep tag (Genscript). The pFastBac plasmid and DH10Bac E. coli (Life Technologies) were used to create recombinant bacmids. The bacmid was transfected into Sf9 cells (Expression Systems) with Cellfectin II (Life Technologies) to generate recombinant baculovirus. The baculovirus was amplified through two passages in Sf9 cells, and then used to infect 1 L of Sf21 cells (Expression Systems) and incubated for 48 hrs at 27°C. Cells were harvested by centrifugation, resuspended . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.18.997585 doi: bioRxiv preprint in wash buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 1 mM MgCl 2 , 5 mM DTT) with 143 µL of BioLock per liter of culture. Cells were lysed via microfluidization (Microfluidics). Lysates were cleared by centrifugation and filtration. The protein was purified using Strep Tactin superflow agarose (IBA). Strep Tactin eluted protein was further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Life Sciences) in 25 mM HEPES, 300 mM NaCl, 100 µM MgCl 2 , 2 mM TCEP, at pH 7.4. Pure protein was concentrated by ultrafiltration prior to flash freezing in liquid nitrogen.