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SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies
Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019–2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restricti...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505060/ https://www.ncbi.nlm.nih.gov/pubmed/36146795 http://dx.doi.org/10.3390/v14091989 |
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author | Plavec, Zlatka Domanska, Aušra Liu, Xiaonan Laine, Pia Paulin, Lars Varjosalo, Markku Auvinen, Petri Wolf, Sharon G. Anastasina, Maria Butcher, Sarah J. |
author_facet | Plavec, Zlatka Domanska, Aušra Liu, Xiaonan Laine, Pia Paulin, Lars Varjosalo, Markku Auvinen, Petri Wolf, Sharon G. Anastasina, Maria Butcher, Sarah J. |
author_sort | Plavec, Zlatka |
collection | PubMed |
description | Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019–2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments. |
format | Online Article Text |
id | pubmed-9505060 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95050602022-09-24 SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies Plavec, Zlatka Domanska, Aušra Liu, Xiaonan Laine, Pia Paulin, Lars Varjosalo, Markku Auvinen, Petri Wolf, Sharon G. Anastasina, Maria Butcher, Sarah J. Viruses Article Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019–2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments. MDPI 2022-09-08 /pmc/articles/PMC9505060/ /pubmed/36146795 http://dx.doi.org/10.3390/v14091989 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Plavec, Zlatka Domanska, Aušra Liu, Xiaonan Laine, Pia Paulin, Lars Varjosalo, Markku Auvinen, Petri Wolf, Sharon G. Anastasina, Maria Butcher, Sarah J. SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title | SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title_full | SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title_fullStr | SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title_full_unstemmed | SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title_short | SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies |
title_sort | sars-cov-2 production, purification methods and uv inactivation for proteomics and structural studies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505060/ https://www.ncbi.nlm.nih.gov/pubmed/36146795 http://dx.doi.org/10.3390/v14091989 |
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