Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.
Journal
ACS omega
ISSN: 2470-1343
Titre abrégé: ACS Omega
Pays: United States
ID NLM: 101691658
Informations de publication
Date de publication:
12 Jan 2021
12 Jan 2021
Historique:
received:
22
07
2020
accepted:
03
11
2020
entrez:
18
1
2021
pubmed:
19
1
2021
medline:
19
1
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 33458462
doi: 10.1021/acsomega.0c03512
pmc: PMC7771249
doi:
Types de publication
Journal Article
Langues
eng
Pagination
85-102Subventions
Organisme : NIGMS NIH HHS
ID : F32 GM128303
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA198095
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI125462
Pays : United States
Organisme : NIGMS NIH HHS
ID : U24 GM129539
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM007288
Pays : United States
Organisme : NIAID NIH HHS
ID : U19 AI142777
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI123654
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM129350
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI132633
Pays : United States
Organisme : NIGMS NIH HHS
ID : P41 GM103310
Pays : United States
Organisme : NIAID NIH HHS
ID : F30 AI150055
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI141367
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI143453
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
© 2020 American Chemical Society.
Déclaration de conflit d'intérêts
The authors declare no competing financial interest.
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