Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

Wanchao Yin; Chunyou Mao; Xiaodong Luan; Dan-Dan Shen; Qingya Shen; Haixia Su; Xiaoxi Wang; Fulai Zhou; Wenfeng Zhao; Minqi Gao; Shenghai Chang; Yuan-Chao Xie; Guanghui Tian; He-Wei Jiang; Sheng-Ce Tao; Jingshan Shen; Yi Jiang; Hualiang Jiang; Yechun Xu; Shuyang Zhang; Yan Zhang; H. Eric Xu

For nsp7 or nsp8 expressed in bacterial, the purification was similar to the purification of nsp7 and nsp8 reported before (15) . Briefly, the resuspended cells were lysed using a high-pressure homogenizer operating at 800 bar. Lysates were cleared by centrifugation at 25,000 × g for 30 min and were then bound to Ni-NTA beads (GE Healthcare). After extend washing with buffer containing 50 mM imidazole, the protein was eluted with buffer containing 300 mM imidazole. The eluted nsp7 sample was concentrated with a 3 kDa molecular weight cut-off Centrifuge Filter (Millipore Corporation) and then size-separated by a Superdex S75 10/300 GL column in 25 mM HEPES pH 7.4, 200 mM sodium chloride, 5% (v/v) glycerol. The eluted nsp8 sample was concentrated with a 30 kDa molecular weight cut-off Centrifuge Filter (Millipore Corporation) and then size-separated by a Superdex S75 10/300 GL column in 25 mM HEPES pH 7.4, 200 mM sodium chloride, 5% (v/v) glycerol. The fractions for the nsp7 or nsp8 were collected, concentrated to about 10 mg/ml, and stored at − 80 °C until use. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.032763 doi: bioRxiv preprint HEPES pH 7.4, 300 mM sodium chloride, 300 mM imidazole, 1 mM magnesium chloride, 1 mM TCEP and 10% (v/v) glycerol.