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

García-Cordero, Julio; Mendoza-Ramírez, Juvenal; Fernández-Benavides, David; Roa-Velazquez, Daniela; Filisola-Villaseñor, Jessica; Martínez-Frías, Sandra Paola; Sanchez-Salguero, Erik Saul; Miguel-Rodríguez, Carlos E.; Maravillas Montero, Jose L.; Torres-Ruiz, Jose J.; Gómez-Martín, Diana; Argumedo, Leopoldo Santos; Morales-Ríos, Edgar; Alvarado-Orozco, Juan M.; Cedillo-Barrón, Leticia

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.