tetano
Editor, Senior Moderator
ACS Omega
. 2020 Dec 21;6(1):85-102.
doi: 10.1021/acsomega.0c03512. eCollection 2021 Jan 12.
Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
Natalia G Herrera[SUP] 1 [/SUP], Nicholas C Morano[SUP] 1 [/SUP], Alev Celikgil[SUP] 1 [/SUP], George I Georgiev[SUP] 1 [/SUP], Ryan J Malonis[SUP] 1 [/SUP], James H Lee[SUP] 1 [/SUP], Karen Tong[SUP] 1 [/SUP], Olivia Vergnolle[SUP] 1 [/SUP], Aldo B Massimi[SUP] 1 [/SUP], Laura Y Yen[SUP] 2 [/SUP], Alex J Noble[SUP] 2 [/SUP], Mykhailo Kopylov[SUP] 2 [/SUP], Jeffrey B Bonanno[SUP] 1 [/SUP], Sarah C Garrett-Thomson[SUP] 1 [/SUP], David B Hayes[SUP] 3 [/SUP], Robert H Bortz 3rd[SUP] 4 [/SUP], Ariel S Wirchnianski[SUP] 1 4 [/SUP], Catalina Florez[SUP] 4 5 [/SUP], Ethan Laudermilch[SUP] 4 [/SUP], Denise Haslwanter[SUP] 4 [/SUP], J Maximilian Fels[SUP] 4 [/SUP], M Eugenia Dieterle[SUP] 4 [/SUP], Rohit K Jangra[SUP] 4 [/SUP], Jason Barnhill[SUP] 5 [/SUP], Amanda Mengotto[SUP] 6 [/SUP], Duncan Kimmel[SUP] 6 [/SUP], Johanna P Daily[SUP] 4 6 [/SUP], Liise-Anne Pirofski[SUP] 4 6 [/SUP], Kartik Chandran[SUP] 4 [/SUP], Michael Brenowitz[SUP] 1 [/SUP], Scott J Garforth[SUP] 1 [/SUP], Edward T Eng[SUP] 2 [/SUP], Jonathan R Lai[SUP] 1 [/SUP], Steven C Almo[SUP] 1 [/SUP]
Affiliations
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.
. 2020 Dec 21;6(1):85-102.
doi: 10.1021/acsomega.0c03512. eCollection 2021 Jan 12.
Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
Natalia G Herrera[SUP] 1 [/SUP], Nicholas C Morano[SUP] 1 [/SUP], Alev Celikgil[SUP] 1 [/SUP], George I Georgiev[SUP] 1 [/SUP], Ryan J Malonis[SUP] 1 [/SUP], James H Lee[SUP] 1 [/SUP], Karen Tong[SUP] 1 [/SUP], Olivia Vergnolle[SUP] 1 [/SUP], Aldo B Massimi[SUP] 1 [/SUP], Laura Y Yen[SUP] 2 [/SUP], Alex J Noble[SUP] 2 [/SUP], Mykhailo Kopylov[SUP] 2 [/SUP], Jeffrey B Bonanno[SUP] 1 [/SUP], Sarah C Garrett-Thomson[SUP] 1 [/SUP], David B Hayes[SUP] 3 [/SUP], Robert H Bortz 3rd[SUP] 4 [/SUP], Ariel S Wirchnianski[SUP] 1 4 [/SUP], Catalina Florez[SUP] 4 5 [/SUP], Ethan Laudermilch[SUP] 4 [/SUP], Denise Haslwanter[SUP] 4 [/SUP], J Maximilian Fels[SUP] 4 [/SUP], M Eugenia Dieterle[SUP] 4 [/SUP], Rohit K Jangra[SUP] 4 [/SUP], Jason Barnhill[SUP] 5 [/SUP], Amanda Mengotto[SUP] 6 [/SUP], Duncan Kimmel[SUP] 6 [/SUP], Johanna P Daily[SUP] 4 6 [/SUP], Liise-Anne Pirofski[SUP] 4 6 [/SUP], Kartik Chandran[SUP] 4 [/SUP], Michael Brenowitz[SUP] 1 [/SUP], Scott J Garforth[SUP] 1 [/SUP], Edward T Eng[SUP] 2 [/SUP], Jonathan R Lai[SUP] 1 [/SUP], Steven C Almo[SUP] 1 [/SUP]
Affiliations
- PMID: 33458462
- PMCID: PMC7771249
- DOI: 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.