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Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

ABSTRACT: The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L^-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

SUBMITTER: Abraham E

PROVIDER: S-EPMC10909970 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

Ábrahám Edit E Bajusz Csaba C Marton Annamária A Borics Attila A Mdluli Thandiswa T Pardi Norbert N Lipinszki Zoltán Z

FEBS open bio 20240124 3

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD fro ...[more]

PMID: 38129177

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Project description:AimSpike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.MethodsThree fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.ResultsRBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively.ConclusionIn this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Dataset Information

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

Publications

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

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