Researchers at the Paul Scherrer Institute in Switzerland have developed a high-quality recombinant virus protein that could be applied in the development of vaccines against the 2019 novel coronavirus (2019-nCoV) and in the detection of novel viruses or antibodies.
The team has developed a soluble 2019-nCoV spike receptor-binding domain (RBD) that is labeled with a fluorescent protein to enable easy detection.
The spike RBD is the subdomain found on 2019-nCoV that binds the human host cell receptor angiotensin-converting enzyme 2 (ACE2) to gain viral entry.
The spike RBD is, therefore, a key target in drug discovery research and vaccine development.
Roger Benoit and colleagues say the fusion protein they have developed is of high protein purity, soluble, and capable of forming a complex with the peptidase domain of human ACE2.
Furthermore, the protein expression and purification protocol the researchers used only requires standard cell culture techniques and equipment.
A pre-print version of the paper is available on the server bioRxiv*, while the article undergoes peer review.
Schematic overview of the key features of the expression region of the construct (not to scale). CMV promoter (PCMV), tetracycline operators (2X TetO2 ), Kozak consensus sequence (Kozak), human interferon alpha 2 (IFNα2) signal peptide including start codon for the complete fusion protein, yellow fluorescent protein (YFP), human rhinovirus 3C protease cleavage site (HRV 3C), 2019-nCoV spike RBD (S RBD), 8xHis-tag (His-tag), two stop codons (Stop), BGH polyadenylation sequence (BGA pA). Important unique restriction enzyme recognition sites are indicated.
Important targets in drug discovery and vaccine development studies
2019-nCoV is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic that continues to sweep the globe, posing a major public health threat.
One crucial area of interest to scientists is the development of high-quality recombinant virus proteins that would aid research related to vaccine development and improved detection assays, as well as an overall understanding of the virus.
Since the RBD of 2019-nCoV binds human ACE2 with high affinity, it has become an important target in drug discovery and vaccine development research.
What did the researchers do?
Benoit and colleagues set out to produce a high-quality, soluble 2019-nCoV spike RBD that is fluorescently labeled to improve detection.
Now the researchers have described a construct and protocol for the expression and purification of milligram amounts of N-terminally yellow fluorescent protein (YFP)-labeled 2019-nCoV spike RBD.
The fusion protein comprises an N-terminal interferon alpha 2 (IFNa2) signal peptide, an enhanced YFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein, and a C-terminal 8x His-tag.
Since the 2019-nCoV spike RBD contains disulfide bonds and N-linked glycosylations, it is typically produced via secretion from eukaryotic cells.
The team reports that expression of the fusion protein is performed via secretion into serum-free medium from adherent, stably transfected human embryonic kidney 293 cells.
“The protocol involves only standard cell culture techniques and equipment,” writes the team.
High protein purity was achieved
Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the Ni-NTA Immobilized Metal Ion Affinity Chromatography (IMAC) purification process the team used yielded very high protein purity.
The initial small scale IMAC purification from 200 µl Ni-NTA resin resulted in 280 µg protein of high purity.
Analysis by size-exclusion chromatography (SEC) and negative staining electron microscopy showed that the fusion protein was soluble and monodisperse.
On incubating the purified fusion protein with the enzyme Peptide: N-glycosidase F (PNGase F), the team found that migration on SDS-PAGE was slightly faster, indicating the presence of N-linked glycosylations in the secreted protein.
Mass spectrometry analysis confirmed the presence of these glycosylations and their reduction following PNGase treatment.
The fusion protein formed a complex with human ACE2
To test whether the YFP-labelled fusion protein binds target human ACE2, the team produced purified human ACE2 peptidase domain and analyzed the two proteins by SEC. The analysis confirmed that the fusion protein binds the human ACE2 peptidase domain, both when fused with YFP and following removal of YFP by proteolytic cleavage.
The findings led the team to propose potential research areas where the fusion protein could be applied.
“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,” conclude Benoit and colleagues.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.