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dc.contributor.author
Bierig, Tobias
dc.contributor.author
Collu, Gabriella
dc.contributor.author
Blanc, Alain
dc.contributor.author
Poghosyan, Emiliya
dc.contributor.author
Benoit, Roger M.
dc.date.accessioned
2021-01-11T08:31:42Z
dc.date.available
2021-01-11T03:51:47Z
dc.date.available
2021-01-11T08:31:42Z
dc.date.issued
2020-12
dc.identifier.issn
2296-4185
dc.identifier.other
10.3389/fbioe.2020.618615
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/460914
dc.identifier.doi
10.3929/ethz-b-000460914
dc.description.abstract
2019-nCoV is the causative agent of the serious, still ongoing, worldwide coronavirus disease (COVID-19) pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease (HRV3C) cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA immobilized metal ion affinity chromatography (IMAC) purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Frontiers Research Foundation
en_US
dc.rights.uri
http://creativecommons.org/licenses/by/4.0/
dc.subject
fluorescent spike RBD fusion protein
en_US
dc.subject
YFP-spike_RBD
en_US
dc.subject
protein reagent for COVID-19 research
en_US
dc.subject
2019- nCoV spike-RBD production using standard cell culture equipment and techniques
en_US
dc.subject
secreted glycosylated YFPlabeled protein
en_US
dc.subject
mammalian protein secretion
en_US
dc.title
Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution 4.0 International
dc.date.published
2020-12-21
ethz.journal.title
Frontiers in Bioengineering and Biotechnology
ethz.journal.volume
8
en_US
ethz.journal.abbreviated
Front. Bioeng. Biotechnol.
ethz.pages.start
618615
en_US
ethz.size
10 p.
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.wos
ethz.identifier.scopus
ethz.publication.place
Lausanne
en_US
ethz.publication.status
published
en_US
ethz.date.deposited
2021-01-11T03:52:08Z
ethz.source
SCOPUS
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2021-01-11T08:31:51Z
ethz.rosetta.lastUpdated
2021-02-15T23:04:21Z
ethz.rosetta.exportRequired
true
ethz.rosetta.versionExported
true
ethz.COinS
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