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

Abstract

Coronavirus disease 2019 ( <b>COVID-19</b> ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( <b>SARS-CoV-2</b> ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( <b>S</b> ) 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 ( <b>cryo-EM</b> ) 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.