tetano
Editor, Senior Moderator
Front Bioeng Biotechnol
. 2020 Dec 21;8:618615.
doi: 10.3389/fbioe.2020.618615. eCollection 2020.
Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct
Tobias Bierig[SUP] 1 2 [/SUP], Gabriella Collu[SUP] 1 2 [/SUP], Alain Blanc[SUP] 3 [/SUP], Emiliya Poghosyan[SUP] 1 [/SUP], Roger M Benoit[SUP] 1 [/SUP]
Affiliations
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.
Keywords: 2019-nCoV spike-RBD production using standard cell culture equipment and techniques; YFP-spike_RBD; fluorescent spike RBD fusion protein; mammalian protein secretion; protein reagent for COVID-19 research; secreted glycosylated YFP-labeled protein.
. 2020 Dec 21;8:618615.
doi: 10.3389/fbioe.2020.618615. eCollection 2020.
Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct
Tobias Bierig[SUP] 1 2 [/SUP], Gabriella Collu[SUP] 1 2 [/SUP], Alain Blanc[SUP] 3 [/SUP], Emiliya Poghosyan[SUP] 1 [/SUP], Roger M Benoit[SUP] 1 [/SUP]
Affiliations
- PMID: 33409271
- PMCID: PMC7779597
- DOI: 10.3389/fbioe.2020.618615
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.
Keywords: 2019-nCoV spike-RBD production using standard cell culture equipment and techniques; YFP-spike_RBD; fluorescent spike RBD fusion protein; mammalian protein secretion; protein reagent for COVID-19 research; secreted glycosylated YFP-labeled protein.