Protocol for production and purification of SARS-CoV-2 3CLpro
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:4 |
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| Enthalten in: |
STAR protocols - 4(2023), 2 vom: 05. Mai, Seite 102326 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Mazzei, Luca [VerfasserIn] |
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| Links: |
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| Themen: |
Journal Article |
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| Anmerkungen: |
Date Revised 14.06.2023 published: Print-Electronic Citation Status Publisher |
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| doi: |
10.1016/j.xpro.2023.102326 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM357377478 |
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| 520 | |a Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Microbiology | |
| 650 | 4 | |a Protein Biochemistry | |
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| 700 | 1 | |a Greene-Cramer, Rebecca |e verfasserin |4 aut | |
| 700 | 1 | |a Bafna, Khushboo |e verfasserin |4 aut | |
| 700 | 1 | |a Jovanovic, Aleksandar |e verfasserin |4 aut | |
| 700 | 1 | |a De Falco, Anna |e verfasserin |4 aut | |
| 700 | 1 | |a Acton, Thomas B |e verfasserin |4 aut | |
| 700 | 1 | |a Royer, Catherine Ann |e verfasserin |4 aut | |
| 700 | 1 | |a Ciurli, Stefano |e verfasserin |4 aut | |
| 700 | 1 | |a Montelione, Gaetano T |e verfasserin |4 aut | |
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