Preparation of SARS-CoV-2 Polymerase Nsp12 and Optimization of Expression Conditions

Abstract

he core component of transcription and replication of SARS-CoV-2 is Nsp12, a non-structural protein that function as an RNA-dependent RNA polymerase. Nsp12 is a key drug target for the development of anti-coronavirus drugs. The purpose of this study was to explore and optimize the expression conditions of Nsp12 of SARS-CoV-2 with the aim of establishing expression conditions to obtain high-purity Nsp12 protein. This study compared the “ice-water bath cooling” and “shaking table cooling” methods and explored the differences in cooling rates and protein expression and properties using the two cooling methods. The shaking table cooling method resulted in reduced amounts of ArnA protein in the Nsp12 protein samples compared with levels from the ice-water bath cooling method. The shaking table method enabled purification of high-quality Nsp12 protein and ensured stable output of the protein. These methods will enable subsequent exploration of the SARS-CoV-2 replication mechanism, the development of specific drugs and drug screening for anti-SARS-CoV-2 pneumonitis.