Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
ABSTRACT 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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Preprint| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
bioRxiv.org - (2022) vom: 28. Okt. Zur Gesamtaufnahme - year:2022 |
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| Sprache: |
Englisch |
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| doi: |
10.1101/2020.06.14.150607 |
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| PPN (Katalog-ID): |
XBI018157831 |
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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: Biophysical, Biochemical, Structural, and Antigenic Analysis |
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| 520 | |a ABSTRACT 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 | |
| 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 | |
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| 700 | 1 | |a Hayes, David B. |e verfasserin |4 aut | |
| 700 | 1 | |a Bortz, Robert H. |e verfasserin |4 aut | |
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