Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.

Induction of high affinity monoclonal antibodies against SARS-CoV-2 variant infection using a DNA prime-protein boost strategy

“…The spike genes from other human coronaviruses, MERS-CoV (accession number: KJ782549.1), human OC43 (accession number: KF572815.1), HKU1 (accession number: DQ437607.1), 229E (accession number: AB691763.1), and NL63 (accession number: KM055633.1), were optimized and synthesized into the pCIneo vector with an HA-tag sequence at the 3’ end of the genes by AllBio Science Inc. The chimeras of SARS-CoV-2 and SARS-CoV-1 were synthesized and subcloned into pcDNA3.1 with a His-tag at the 3’ end of genes, as previously described[ 29 ]. All plasmids were transformed into DH5α E. coli for plasmid amplification.…”

Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System

“…The cytoplasm of Escherichia coli is under reducing conditions, and consequently RBD aggregates in the inclusion body [ 10 ]. Mammalian cell expression systems can synthesize proteins with precise PTMs and proper folding containing disulfide bonds, but require prolonged incubation times of 5–7 days or longer [ 12 ]. The WG cell-free protein expression system is considered as a simple and efficient alternative to the mammalian cell system but produces RBD devoid of S-S, which is essential for its biological activity.…”

“…Insect-based systems involve a cumbersome procedure of baculovirus vector production [ 11 ]. Mammalian cell-based systems can produce excellently folded proteins with precise PTMs but have the disadvantage of demanding culture conditions and requiring long culture times as compared with prokaryotic and cell-free systems [ 12 ].…”

Antibodies Produced Toward Recombinant RBD and Nucleocapsid Neutralize SARS-COV-2

“…Recombinant RBD and N proteins were expressed in the bacterial host at approximately 40 mg/L for RBD and 50 mg/L for N protein. The average expression of RBD and N in eukaryotic hosts are 45 mg/L and 50 mg/L, respectively 44 – 48 . Despite the glycosylation in eukaryotic cells and the sugar removal process, it seems appropriate for expressing these proteins in the bacterial host.…”