Recombinant RBD of the SARS-CoV-2 Spike Protein: Production in Escherichia coli Cells, Binding to Antibodies, and Antiviral Activity

• Published in: Molecular genetics, microbiology and virology microbiology and virology, 2023-06, Vol.38 (2), p.86-94
• Main Authors: Gromova, M. S., Gromov, A. V., Grunina, T. M., Lyashchuk, A. M., Galushkina, Z. M., Subbotina, M. E., Esmagambetov, I. B., Ryabova, E. I., Prokofiev, V. V., Kovyrshina, A. V., Ilyukhina, A. A., Shelkov, A. Y., Karyagina, A. S., Lunin, V. G.
• Format: Article
• Language: English
• Subjects: ACE2; Angiotensin-converting enzyme 2; Antibodies; Antiviral activity; Biomedical and Life Sciences; Chromatography; E coli; Enzyme immunoassay; Enzymes; Escherichia coli; Experimental Papers; Immunoassay; Life Sciences; Microbiology; Molecular Medicine; Monoclonal antibodies; Protein purification; Proteins; Severe acute respiratory syndrome coronavirus 2; Spike protein; Viruses
• ISSN: 0891-4168; 1934-841X
• DOI: 10.3103/S0891416823020052

The aim of the study is to synthesize a recombinant protein in Escherichia coli cells that carries the receptor-binding domain (RBD) of Spike protein of SARS CoV-2, with antiviral activity comparable to the activity of RBD obtained in a eukaryotic cells (eRBD). 6His-RBD short (24.4 kDa) and 6His-RBD long (33.7 kDa) proteins were expressed in Escherichia coli strain BL21 (DE3). Chromatographic purification of the proteins was carried out on WorkBeads 40 Ni-NTA and WorkBeads 40S sorbents followed by multistage refolding. Enzyme immunoassay was performed using GamP2C5 and GamXRH19 humanized single-domain monoclonal antibodies specific for SARS-CoV-2 Spike protein RBD. Antiviral activity against the SARS-CoV-2 virus was studied using Vero E6 cells. 6His-RBD short recombinant protein was synthesized in Escherichia coli cells, including the Spike protein RBM (receptor-binding motif) of SARS-CoV-2 virus (330–527 а.а.). Two-stage chromatographic purification of 6His-RBD short recombinant protein was performed, followed by refolding. Enzyme immunoassay demonstrated effective interaction of 6His-RBD short recombinant protein with virus neutralizing antibodies, comparable to eRBD. The study of antiviral activity showed inhibition of SARS-CoV-2 virus reproduction after treatment of Vero E6 cells with 6His-RBD short (45.1%) and eRBD (42.8%) proteins. The 6His-RBD long recombinant protein obtained in the same work, which included a longer fragment of RBD, did not interact with virus neutralizing antibodies and did not inhibit the replication of the SARS-CoV-2 virus. After conducting additional studies, the developed 6His-RBD short recombinant protein can be considered a promising drug for therapeutic use as an ACE2 receptor blocker.