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Updated: Nov 7, 2025
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli
Clément Madru1, Ayten Dizkirici Tekpinar1,2, Sandrine Rosario1
1Unit of Structural Dynamics of Macromolecules, Institut Pasteur & CNRS UMR, Paris, France.
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).
Insights
A new method co-expresses the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex in E. coli. This rapid purification of functional RdRp accelerates antiviral drug discovery for COVID-19.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes COVID-19, necessitating new antiviral treatments.
- SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is crucial for viral replication and a key antiviral target.
- Current methods for producing RdRp involve separate subunit purification and in vitro assembly, facing challenges like nsp12 solubility.
Purpose of the Study:
- To develop an improved strategy for producing the complete SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex.
- To facilitate rapid screening of antiviral compounds against SARS-CoV-2 RdRp.
- To provide a readily accessible tool for the scientific community.
Main Methods:
- Co-expression of SARS-CoV-2 nsp7, nsp8, and nsp12 subunits using a single plasmid in E. coli.
- Purification of the recombinant RdRp complex.
- Characterization of the complex stoichiometry and RNA polymerization activity via primer-extension assays.
Main Results:
- Successful co-expression and purification of the functional SARS-CoV-2 RdRp complex with (nsp7)(nsp8)2(nsp12) stoichiometry.
- Demonstration of RNA polymerization activity by the purified enzyme.
- A significantly expedited purification protocol, achievable within one day.
Conclusions:
- The developed single-plasmid co-expression system offers a fast and efficient method for obtaining functional SARS-CoV-2 RdRp in E. coli.
- This approach overcomes previous limitations in RdRp production and is suitable for high-throughput screening.
- The plasmid has been shared via Addgene (ID: 165451) to support antiviral research efforts.
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