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.

• Authors: Natalia G Herrera, Nicholas C Morano, Alev Celikgil, George I Georgiev, Ryan J Malonis, James H Lee, Karen Tong, Olivia Vergnolle, Aldo B Massimi, Laura Y Yen, Alex J Noble, Mykhailo Kopylov, Jeffrey B Bonanno, Sarah C Garrett-Thomson, David B Hayes, Robert H Bortz 3rd, Ariel S Wirchnianski, Catalina Florez, Ethan Laudermilch, Denise Haslwanter, J Maximilian Fels, M Eugenia Dieterle, Rohit K Jangra, Jason Barnhill, Amanda Mengotto, Duncan Kimmel, Johanna P Daily, Liise-Anne Pirofski, Kartik Chandran, Michael Brenowitz, Scott J Garforth, Edward T Eng, Jonathan R Lai, Steven C Almo
• Journal: bioRxiv : the preprint server for biology (bioRxiv) (Jun 17 2020) United States
• PMID: 32587972 (Publication Type: Preprint)