Paper

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

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

2020

Overview

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 Expi293FTM and ExpiCHO-STM cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-STM 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. Competing Interest Statement The authors have declared no competing interest. Footnotes * Additional Authors and Methods were added for the collection and curation of serum, and the development of the ELISA assay used in this paper.

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Antigenicity / Biochemistry / Cell lines / Coronaviruses / COVID-19 / Enzyme-linked immunosorbent assay / Glycoproteins / Pandemics / Protein arrays / Protein purification / Proteins / Secretome / Serology / Severe acute respiratory syndrome coronavirus 2