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bioRxiv · 10.1101/2020.06.14.150607

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Herrera, N. G.·Morano, N. C.·Celikgil, A.·Georgiev, G. I.·Malonis, R. J.·Lee, J. H.·Tong, K.·Vergnolle, O.·Massimi, A. B.·Yen, L. Y.·Noble, A. J.·Kopylov, M.·Bonanno, J. B.·Garrett-Thompson, S. C.·Hayes, D. B.·Brenowitz, M.·Garforth, S. J.·Eng, E. T.·Lai, J. R.·Almo, S. C.
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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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Herrera, N. G., Morano, N. C., Celikgil, A., Georgiev, G. I., Malonis, R. J., Lee, J. H., Tong, K., Vergnolle, O., Massimi, A. B., Yen, L. Y., Noble, A. J., Kopylov, M., Bonanno, J. B., Garrett-Thompson, S. C., Hayes, D. B., Brenowitz, M., Garforth, S. J., Eng, E. T., Lai, J. R., Almo, S. C.. 2020-06-15. Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis. https://doi.org/10.1101/2020.06.14.150607

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