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
Mazzei, L., Greene-Cramer, R., Bafna, K., Jovanovic, A., De Falco, A., Acton, T.B., et al. (2023). Protocol for production and purification of SARS-CoV-2 3CLpro. STAR PROTOCOLS, 4(2), 1-30 [10.1016/j.xpro.2023.102326].
Protocol for production and purification of SARS-CoV-2 3CLpro
Mazzei L.;Ciurli S.
;
2023
Abstract
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| File | Dimensione | Formato | |
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Mazzei-Protocol for production and purification of SARS-CoV-2 3CLpro-2023-STAR Protocols.pdf
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