Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct.

Tobias Bierig,Gabriella Collu,Roger M Benoit,Emiliya Poghosyan,Alain Blanc

doi:10.3389/fbioe.2020.618615

Tobias Bierig, Gabriella Collu + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2020.618615

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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.

Highlights

The membrane-anchored, trimeric spike (S) glycoproteins are the most prominent protrusions on the surface of the novel coronavirus (2019-nCoV) (Figure 1A)
Our aim was to produce high-quality, soluble 2019-nCoV spike receptor-binding domain (RBD) labeled with a fluorescent protein for easy detection
As in the construct described by Román et al (2016), we placed the signal peptide directly upstream of the fluorescent protein, we left out the start methionine of yellow fluorescent protein (YFP), since translation starts at the start ATG of the signal peptide upstream of the YFP

Summary

Introduction

The membrane-anchored, trimeric spike (S) glycoproteins are the most prominent protrusions on the surface of the novel coronavirus (2019-nCoV) (Figure 1A). Coronavirus (CoV) spike proteins typically comprise two subunits (Figure 1B). The S1 subunit is responsible for receptor binding and the S2 subunit is involved in fusing the membranes of the virus and the host (Li, 2016). The S1 subunit is composed of an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD) (Figure 1C; Li, 2016). S1-CTD comprises two sub-domains, one functioning as a core structure, the other one as a receptor-binding motif (Figure 1C; Li, 2016). The receptor-binding domain of 2019nCoV binds human angiotensin-converting enzyme 2 (ACE2) with high affinity (Wrapp et al, 2020).

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