Characterization of the SARS-CoV-2 S Protein : Biophysical, Biochemical, Structural, and Antigenic Analysis
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 expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). 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. 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.
| Errataetall: |
UpdateIn: ACS Omega. 2020 Dec 21;6(1):85-102. - PMID 33458462 |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2020 |
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| Enthalten in: |
bioRxiv : the preprint server for biology - (2020) vom: 17. Juni |
| Sprache: |
Englisch |
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| Anmerkungen: |
Date Revised 29.03.2024 published: Electronic UpdateIn: ACS Omega. 2020 Dec 21;6(1):85-102. - PMID 33458462 Citation Status PubMed-not-MEDLINE |
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| doi: |
10.1101/2020.06.14.150607 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM311662552 |
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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 |
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| 500 | |a Date Revised 29.03.2024 | ||
| 500 | |a published: Electronic | ||
| 500 | |a UpdateIn: ACS Omega. 2020 Dec 21;6(1):85-102. - PMID 33458462 | ||
| 500 | |a Citation Status PubMed-not-MEDLINE | ||
| 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 expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). 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. 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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| 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 | |
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