Expression and characterization of SARS-CoV-2 spike proteins.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
received:
21
12
2020
accepted:
06
09
2021
pubmed:
7
10
2021
medline:
11
11
2021
entrez:
6
10
2021
Statut:
ppublish
Résumé
The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.
Identifiants
pubmed: 34611365
doi: 10.1038/s41596-021-00623-0
pii: 10.1038/s41596-021-00623-0
pmc: PMC9665560
mid: NIHMS1846725
doi:
Substances chimiques
Spike Glycoprotein, Coronavirus
0
spike protein, SARS-CoV-2
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
5339-5356Subventions
Organisme : NIAID NIH HHS
ID : T32 AI007392
Pays : United States
Organisme : NCI NIH HHS
ID : HHSN261200800001E
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM124141
Pays : United States
Organisme : Bill & Melinda Gates Foundation
ID : INV-017592
Pays : United States
Organisme : U.S. Department of Health & Human Services | National Institutes of Health (NIH)
ID : AI127521
Organisme : Welch Foundation
ID : F-1767
Organisme : National Science Foundation (NSF)
ID : 1453358
Organisme : NIGMS NIH HHS
ID : R01 GM120554
Pays : United States
Organisme : Welch Foundation
ID : F-1808
Organisme : NIAID NIH HHS
ID : R01 AI127521
Pays : United States
Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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