Characterization of the SARS-CoV-2 S Protein : Biophysical, Biochemical, Structural, and Antigenic Analysis
© 2020 American Chemical Society..
Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:6 |
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| Enthalten in: |
ACS omega - 6(2021), 1 vom: 12. Jan., Seite 85-102 |
| Sprache: |
Englisch |
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| Anmerkungen: |
Date Revised 29.08.2023 published: Electronic-eCollection UpdateOf: bioRxiv. 2020 Jun 17;:. - PMID 32587972 Citation Status PubMed-not-MEDLINE |
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| doi: |
10.1021/acsomega.0c03512 |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM320210723 |
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| 100 | 1 | |a Herrera, Natalia G |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Characterization of the SARS-CoV-2 S Protein |b Biophysical, Biochemical, Structural, and Antigenic Analysis |
| 264 | 1 | |c 2021 | |
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| 500 | |a Date Revised 29.08.2023 | ||
| 500 | |a published: Electronic-eCollection | ||
| 500 | |a UpdateOf: bioRxiv. 2020 Jun 17;:. - PMID 32587972 | ||
| 500 | |a Citation Status PubMed-not-MEDLINE | ||
| 520 | |a © 2020 American Chemical Society. | ||
| 520 | |a Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2 | ||
| 650 | 4 | |a Journal Article | |
| 700 | 1 | |a Morano, Nicholas C |e verfasserin |4 aut | |
| 700 | 1 | |a Celikgil, Alev |e verfasserin |4 aut | |
| 700 | 1 | |a Georgiev, George I |e verfasserin |4 aut | |
| 700 | 1 | |a Malonis, Ryan J |e verfasserin |4 aut | |
| 700 | 1 | |a Lee, James H |e verfasserin |4 aut | |
| 700 | 1 | |a Tong, Karen |e verfasserin |4 aut | |
| 700 | 1 | |a Vergnolle, Olivia |e verfasserin |4 aut | |
| 700 | 1 | |a Massimi, Aldo B |e verfasserin |4 aut | |
| 700 | 1 | |a Yen, Laura Y |e verfasserin |4 aut | |
| 700 | 1 | |a Noble, Alex J |e verfasserin |4 aut | |
| 700 | 1 | |a Kopylov, Mykhailo |e verfasserin |4 aut | |
| 700 | 1 | |a Bonanno, Jeffrey B |e verfasserin |4 aut | |
| 700 | 1 | |a Garrett-Thomson, Sarah C |e verfasserin |4 aut | |
| 700 | 1 | |a Hayes, David B |e verfasserin |4 aut | |
| 700 | 1 | |a Bortz, Robert H |c 3rd |e verfasserin |4 aut | |
| 700 | 1 | |a Wirchnianski, Ariel S |e verfasserin |4 aut | |
| 700 | 1 | |a Florez, Catalina |e verfasserin |4 aut | |
| 700 | 1 | |a Laudermilch, Ethan |e verfasserin |4 aut | |
| 700 | 1 | |a Haslwanter, Denise |e verfasserin |4 aut | |
| 700 | 1 | |a Fels, J Maximilian |e verfasserin |4 aut | |
| 700 | 1 | |a Dieterle, M Eugenia |e verfasserin |4 aut | |
| 700 | 1 | |a Jangra, Rohit K |e verfasserin |4 aut | |
| 700 | 1 | |a Barnhill, Jason |e verfasserin |4 aut | |
| 700 | 1 | |a Mengotto, Amanda |e verfasserin |4 aut | |
| 700 | 1 | |a Kimmel, Duncan |e verfasserin |4 aut | |
| 700 | 1 | |a Daily, Johanna P |e verfasserin |4 aut | |
| 700 | 1 | |a Pirofski, Liise-Anne |e verfasserin |4 aut | |
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| 700 | 1 | |a Almo, Steven C |e verfasserin |4 aut | |
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