Abstract
To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).
Highlights
The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected millions of people with a death toll exceeding two million worldwide [1,2,3,4]
The nsp7, nsp8 and nsp12 genes were optimized for recombinant expression in E. coli, and inserted into a modified pRSFDuet-1 vector, allowing the expression of 14 histidine (14his) N-fused proteins
The purified complex was functionally active but partially proteolyzed, exhibiting a fourth band on SDS-PAGE analysis after four purification steps (S3 Fig in S1 File). This additional band corresponding to an approximately 12 kDa protein was identified as the globular C-terminal part of nsp8, showing that the N-terminal region of nsp8 (~80 amino-acids) is degraded during the purification process
Summary
The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected millions of people with a death toll exceeding two million worldwide [1,2,3,4]. The post-doctoral fellowship of C.M is funded by the Pasteur-Roux-Cantarini fellowship from the Institut Pasteur. While nsp and nsp express readily in Escherichia coli, nsp shows limited solubility in bacterial expression systems and is often produced in insect cells [22, 23, 25,26,27] These approaches multiply the protein expression and purification steps, making RdRp isolation cost- and time-consuming. RNA polymerization activity was measured using primerextension assays and showed that the purified enzyme is functional This approach provides a useful alternative to more expensive and complicated protein expression systems, and offers many practical advantages inherent to bacterial systems, such as easy generation of mutants and simple cultivation handling. Single-day purification protocol results in a stable and active complex that can be used in most protein biochemistry laboratories for drug screening as well as for functional studies
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