Expression and purification of tag-free SARS-CoV-2 RNA-dependent RNA polymerase in Escherichia coli.

The RNA-dependent-RNA polymerase (RdRp) from SARS-CoV-2 is an important drug target because it is responsible for viral RNA genome replication. Efficient production of recombinant RdRp is important in screening antivirals to treat COVID-19. Here, we present our protocol for expression of tag-free replication complex proteins in E. coli and subsequent purification. Despite the added complexity of multiple purification steps, our methods provide greater activity, yield at lower cost, and are faster than baculovirus expression systems. For complete details on the use and execution of this protocol, please refer to Dangerfield et al. (2020).

Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp). However, the enzymatic mechanism and efficiency of Remdesivir have not been determined, and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in E. coli, followed by purification and kinetic analysis of an untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast (k cat  = 240-680 s-1) and processive (k off  = 0.013 s-1) RNA polymerization. The specificity constant (k cat /K m ) for Remdesivir triphosphate (RTP) incorporation (1.29 µM-1s-1) is higher than that for the competing ATP (0.74 µM-1 s-1). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the standard for development of informative enzyme assays to screen for new inhibitors.