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

  • STAR Protoc. 2023 Jun 16;4(2):102326. doi: 10.1016/j.xpro.2023.102326.
Luca Mazzei  1 ,  Rebecca Greene-Cramer  2 ,  Khushboo Bafna  2 ,  Aleksandar Jovanovic  1 ,  Anna De Falco  2 ,  Thomas B Acton  2 ,  Catherine Ann Royer  3 ,  Stefano Ciurli  4 ,  Gaetano T Montelione  5
Affiliations
  • 1. Department of Pharmacy and Biotechnology, University of Bologna, 40127 Bologna, Italy.
  • 2. Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
  • 3. Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
  • 4. Department of Pharmacy and Biotechnology, University of Bologna, 40127 Bologna, Italy. Electronic address: [email protected].
  • 5. Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: [email protected].
PMID: 37235475 DOI: 10.1016/j.xpro.2023.102326
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.

Keywords
Microbiology; Protein Biochemistry; Structural Biology.
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