Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
- 21 December 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in ACS Omega
- Vol. 6 (1), 85-102
- https://doi.org/10.1021/acsomega.0c03512
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 protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614–642 and 828–854. 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 and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. 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.Funding Information
- National Cancer Institute (R01 CA198095)
- National Institute of Allergy and Infectious Diseases (R01AI123654, R01AI125462, R01AI132633, R01AI143453, R21AI141367, U19AI142777)
- G. Harold and Leila Y. Mathers Charitable Foundation
- Pew Charitable Trusts
- National Institute of General Medical Sciences (5R01GM129350, F32GM128303)
This publication has 72 references indexed in Scilit:
- The Phenix software for automated determination of macromolecular structuresMethods, 2011
- Appion: An integrated, database-driven pipeline to facilitate EM image processingJournal of Structural Biology, 2009
- Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with ReceptorScience, 2005
- Automated molecular microscopy: The new Leginon systemJournal of Structural Biology, 2005
- Foldon, The Natural Trimerization Domain of T4 Fibritin, Dissociates into a Monomeric A-state Form containing a Stable β-Hairpin: Atomic Details of Trimer Dissociation and Local β-Hairpin Stability from Residual Dipolar CouplingsJournal of Molecular Biology, 2004
- N-Terminal Domain of the Murine Coronavirus Receptor CEACAM1 Is Responsible for Fusogenic Activation and Conformational Changes of the Spike ProteinJournal of Virology, 2004
- Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirusNature, 2003
- Predicting transmembrane protein topology with a hidden markov model: application to complete genomesJournal of Molecular Biology, 2001
- Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation ModelingBiophysical Journal, 2000
- Efficient selection for high-expression transfectants with a novel eukaryotic vectorGene, 1991