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
Protocol for production and purification of SARS-CoV-2 3CLpro / Mazzei L.; Greene-Cramer R.; Bafna K.; Jovanovic A.; De Falco A.; Acton T.B.; Royer C.A.; Ciurli S.; Montelione G.T.. - In: STAR PROTOCOLS. - ISSN 2666-1667. - STAMPA. - 4:2(2023), pp. 102326.1-102326.30. [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.1I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
