Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRP) is essential for viral replication.
• Expression and purification of the active SARS-CoV-2 RdRP complex (nsp7, nsp8, and nsp12) present significant challenges due to protein folding and complex assembly.
• Previous methods have limitations in providing sufficient quantities of active RdRP for detailed studies.

Purpose of the Study:

• To develop an optimized and reliable protocol for the expression and purification of the active SARS-CoV-2 core RdRP complex.
• To provide a consistent source of milligram quantities of the enzymatically active polymerase complex for further research.
• To facilitate structure, function, and inhibition studies of the SARS-CoV-2 RdRP.

Main Methods:

• Adapted a published method for expressing the core SARS-CoV-2 RdRP complex in Escherichia coli.
• Incorporated a procedure for expressing nsp12 fused to maltose-binding protein in insect cells to enhance complex assembly.
• Utilized affinity tag removal post-purification to obtain the active core polymerase complex with defined stoichiometry.

Main Results:

• Successfully developed a protocol yielding milligram amounts of the active SARS-CoV-2 core RdRP complex.
• Achieved the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12 in the purified complex.
• The protocol provides a reliable platform, overcoming limitations of previous methods.

Conclusions:

• The optimized protocol offers a dependable method for producing the SARS-CoV-2 RdRP complex.
• This reliable source of active polymerase complex is crucial for advancing research into SARS-CoV-2 structure, function, and antiviral drug development.
• Recombinant plasmid constructs are available via Addgene, promoting accessibility for the scientific community.