Functional expression of five refolded recombinant variants of RBD from SARS-CoV-2 in Escherichia coli.

Production of the receptor binding domain, RBD, variants of SARS-CoV-2, has been necessary to understand their interaction with the human receptor ACE2 and to develop alternative strategies against COVID-19. Affordable bacterial production can aid in these efforts and offer alternatives to address the ongoing emergence of new variants. Expressing recombinant RBD in E. coli could provide a valuable alternative if the challenges related to disulfide bond formation, low solubility, and the absence of glycosylation capabilities are addressed in this heterologous expression system. In this study, five representative RBD variants were expressed in E. coli BL21 (two versions of the RBD Wuhan sequence (Wt), Delta, Omicron BA.2, and Omicron JN.1). The resulting inclusion bodies were solubilized using guanidine chloride and refolded in the presence of a buffer containing the redox couple GSH-GSSG and L-arginine monohydrochloride. Purification was achieved through size-exclusion chromatography. These purified RBD variants were analyzed and compared to the RBD with the Wuhan sequence and the Omicron BA.5 variant, both produced in Chinese Hamster Ovary cells, regarding their ability to bind to ACE2 and using surface plasmon resonance, SPR, studies. The strategy outlined in this work did not include expression studies or a complete comparison of the expression yields obtained in relation to other expression systems. However, it clearly demonstrates the feasibility of obtaining various RBD variants, capable of binding to ACE2, starting from inclusion bodies, and exhibiting comparable affinities to those RBD variants produced in eukaryotic cells. This approach provides an additional option to enhance the existing tools available for research laboratories studying interactions between biologically active RBD variants and ACE2.