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

Authors :: Mazzei L
Greene-Cramer R
Bafna K
Jovanovic A
De Falco A
Acton TB
Royer CA
Ciurli S
Montelione GT
Source :: STAR protocols [STAR Protoc] 2023 May 05; Vol. 4 (2), pp. 102326. Date of Electronic Publication: 2023 May 05.
Publication Year :: 2023
Publisher :: Ahead of Print
Abstract: 3CL <superscript>pro</superscript> protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CL <superscript>pro</superscript> production in Escherichia coli. We describe steps to purify 3CL <superscript>pro</superscript> , expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L <superscript>-1</superscript> following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CL <superscript>pro</superscript> 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. <superscript>1</superscript> .<br />Competing Interests: Declaration of interests G.T.M. is a founder and advisor to Nexomics Biosciences, Inc. This does not represent a conflict of interest with respect to this study.<br /> (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)