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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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
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author Mazzei, Luca
Greene-Cramer, Rebecca
Bafna, Khushboo
Jovanovic, Aleksandar
De Falco, Anna
Acton, Thomas B.
Royer, Catherine Ann
Ciurli, Stefano
Montelione, Gaetano T.
author_facet Mazzei, Luca
Greene-Cramer, Rebecca
Bafna, Khushboo
Jovanovic, Aleksandar
De Falco, Anna
Acton, Thomas B.
Royer, Catherine Ann
Ciurli, Stefano
Montelione, Gaetano T.
author_sort Mazzei, Luca
collection PubMed
description 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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spelling pubmed-101605262023-05-05 Protocol for production and purification of SARS-CoV-2 3CL(pro) Mazzei, Luca Greene-Cramer, Rebecca Bafna, Khushboo Jovanovic, Aleksandar De Falco, Anna Acton, Thomas B. Royer, Catherine Ann Ciurli, Stefano Montelione, Gaetano T. STAR Protoc Protocol 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) Elsevier 2023-05-05 /pmc/articles/PMC10160526/ /pubmed/37235475 http://dx.doi.org/10.1016/j.xpro.2023.102326 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Mazzei, Luca
Greene-Cramer, Rebecca
Bafna, Khushboo
Jovanovic, Aleksandar
De Falco, Anna
Acton, Thomas B.
Royer, Catherine Ann
Ciurli, Stefano
Montelione, Gaetano T.
Protocol for production and purification of SARS-CoV-2 3CL(pro)
title Protocol for production and purification of SARS-CoV-2 3CL(pro)
title_full Protocol for production and purification of SARS-CoV-2 3CL(pro)
title_fullStr Protocol for production and purification of SARS-CoV-2 3CL(pro)
title_full_unstemmed Protocol for production and purification of SARS-CoV-2 3CL(pro)
title_short Protocol for production and purification of SARS-CoV-2 3CL(pro)
title_sort protocol for production and purification of sars-cov-2 3cl(pro)
topic Protocol
url 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
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