Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target

Yan Gao; Liming Yan; Yucen Huang; Fengjiang Liu; Yao Zhao; Lin Cao; Tao Wang; Qianqian Sun; Zhenhua Ming; Lianqi Zhang; Ji Ge; Litao Zheng; Ying Zhang; Haofeng Wang; Yan Zhu; Chen Zhu; Tianyu Hu; Tian Hua; Bing Zhang; Xiuna Yang; Jun Li; Haitao Yang; Zhijie Liu; Wenqing Xu; Luke W. Guddat; Quan Wang; Zhiyong Lou; Zihe Rao

The 2019-nCoV nsp12 (GenBank: MN908947) gene was cloned into a modified pET-22a 5 vector, with the C-terminus possessing a 10 × His-tag. The plasmids were transformed into E. coil BL21 (DE3), and the transformed cells were cultured at 37 °C in LB media containing 100 mg/L ampicillin. After the OD600 reached 0.8, the culture was cooled to 16 °C and supplemented with 0.5 mM IPTG. After overnight induction, the cells were harvested through centrifugation, and the pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl, 10 10% glycerol) and homogenized with an ultra-high-pressure cell disrupter at 4 °C. The insoluble material was removed through centrifugation at 12,000 rpm. The fusion protein was first purified by Ni-NTA affinity chromatography and elution with lysis buffer and then further purified by passage through a Hitrap Q ion-exchange column (GE Healthcare, USA) before loading onto a Superdex 200 10/300 Increase column (GE Healthcare, USA) in a buffer containing 20 mM Tris-