Protocol for production and purification of SARS-CoV-2 3CL^pro.

ABSTRACT: 3CL^pro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CL^pro production in Escherichia coli. We describe steps to purify 3CL^pro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L^-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CL^pro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.¹.

SUBMITTER: Mazzei L 

PROVIDER: S-EPMC10160526 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature