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Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain
Tobias Bierig1,2, Gabriella Collu1,2, Alain Blanc3
1Laboratory of Nanoscale Biology, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland.
Frontiers in Bioengineering and Biotechnology
|January 7, 2021
Summary
Researchers developed a method to produce a fluorescently labeled SARS-CoV-2 spike protein receptor-binding domain (RBD). This recombinant protein is crucial for developing COVID-19 vaccines and diagnostic assays.
Area of Science:
- Virology
- Protein Biochemistry
- Molecular Biology
Background:
- The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing COVID-19 pandemic.
- High-quality recombinant SARS-CoV-2 proteins are essential for vaccine development, diagnostic assay improvement, and understanding viral mechanisms.
- The spike (S) protein's receptor-binding domain (RBD) requires specific expression strategies due to its disulfide bonds and glycosylations.
Purpose of the Study:
- To describe a protocol for expressing and purifying a yellow fluorescent protein (YFP)-labeled SARS-CoV-2 spike RBD.
- To create a versatile tool for research into SARS-CoV-2 and the development of countermeasures.
Main Methods:
- A fusion protein construct was designed, including an N-terminal interferon alpha 2 (IFNα2) signal peptide, eYFP, FLAG-tag, human rhinovirus 3C protease (HRV3C) cleavage site, SARS-CoV-2 RBD, and a C-terminal 8x His-tag.
- HEK 293 cells were stably transfected, and the fusion protein was secreted into serum-free medium.
- Purification was achieved using Ni-NTA immobilized metal ion affinity chromatography (IMAC), followed by characterization using SDS-PAGE, size-exclusion chromatography (SEC), and negative staining electron microscopy.
Main Results:
- Highly pure, soluble, and monodisperse YFP-labeled SARS-CoV-2 spike RBD was successfully produced.
- Deglycosylation experiments confirmed the presence of N-linked glycosylations.
- Complex formation with angiotensin-converting enzyme 2 (ACE2), the viral receptor, was verified via SEC for both the fused and cleaved RBD.
Conclusions:
- The developed protocol yields a high-quality, fluorescently labeled SARS-CoV-2 spike RBD.
- This recombinant protein is suitable for various applications, including binding studies, cell imaging, antigen development, and assay creation.
- The YFP-labeled RBD facilitates research and development efforts targeting the COVID-19 pandemic.
