Protocol for production and purification of SARS-CoV-2 3CLpro
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Updated: Oct 23, 2026

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection
Published on: May 2, 2011
Luca Mazzei1, Rebecca Greene-Cramer2, Khushboo Bafna2
1Department of Pharmacy and Biotechnology, University of Bologna, 40127 Bologna, Italy.
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.
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