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

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

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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