Protocol for production and purification of SARS-CoV-2 3CLpro

• Published in: STAR protocols 2023-06, Vol.4 (2), p.102326-102326, Article 102326
• Main Authors: Mazzei, Luca, Greene-Cramer, Rebecca, Bafna, Khushboo, Jovanovic, Aleksandar, De Falco, Anna, Acton, Thomas B., Royer, Catherine Ann, Ciurli, Stefano, Montelione, Gaetano T.
• Format: Article
• Language: English
• Subjects: Microbiology; Protein Biochemistry; Structural Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102326

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, 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 3CLpro 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 [Display omitted] •Production and purification of SARS-CoV-2 3CLprowith native N- and C-termini•3CLproinhibition assay and description for data processing/analysis•Structural characterization by X-ray crystallography and NMR spectroscopy•Detailed troubleshooting and alternative approaches Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, 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 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay.