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

3CL(pro) protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CL(pro) production in Escherichia coli. We describe steps to purify 3CL(pro), expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg...

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Detalles Bibliográficos
Autores principales: Mazzei, Luca, Greene-Cramer, Rebecca, Bafna, Khushboo, Jovanovic, Aleksandar, De Falco, Anna, Acton, Thomas B., Royer, Catherine Ann, Ciurli, Stefano, Montelione, Gaetano T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10160526/
https://www.ncbi.nlm.nih.gov/pubmed/37235475
http://dx.doi.org/10.1016/j.xpro.2023.102326
Descripción
Sumario:3CL(pro) protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CL(pro) production in Escherichia coli. We describe steps to purify 3CL(pro), 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 3CL(pro) 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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