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

Herrera, Natalia G.; Morano, Nicholas C.; Celikgil, Alev; Georgiev, George I.; Malonis, Ryan J.; Lee, James H.; Tong, Karen; Vergnolle, Olivia; Massimi, Aldo B.; Yen, Laura Y.; Noble, Alex J.; Kopylov, Mykhailo; Bonanno, Jeffrey B.; Garrett-Thomson, Sarah C.; Hayes, David B.; Bortz, Robert H.; Wirchnianski, Ariel S.; Florez, Catalina; Laudermilch, Ethan; Haslwanter, Denise; Fels, J. Maximilian; Dieterle, M. Eugenia; Jangra, Rohit K.; Barnhill, Jason; Mengotto, Amanda; Kimmel, Duncan; Daily, Johanna P.; Pirofski, Liise-anne; Chandran, Kartik; Brenowitz, Michael; Garforth, Scott J.; Eng, Edward T.; Lai, Jonathan R.; Almo, Steven C.

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 Expi293Fâ„¢ and ExpiCHO-Sâ„¢ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-Sâ„¢ 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.