Abstract
SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.
Keywords: E484K mutation; RBD; SARS-CoV-2; chaperone; disulphide bond; endoplasmic reticulum; mammalian cells; misfolding; protein disulphide isomerase; protein yield.
【저자키워드】 SARS-CoV-2, Endoplasmic reticulum, RBD, E484K mutation, mammalian cells, chaperone, protein yield., protein disulphide isomerase, misfolding, disulphide bond, 【초록키워드】 COVID-19, Vaccine, Mutation, B.1.351, SARS-CoV-2 variant, diagnostic, diagnostics, variants, Protein, cells, unfolded protein response, E484K, mutant, RBD protein, protocols, B.1.351 variant, accumulation, secretion, domain, effort, upregulation, chaperones, mammalian, recombinant RBD protein, UPR, RBD mutation, resulting, reduced, contribute, expressed, correlated, the RBD, interfere, producing, assist, PDIA2, RBD-Fc fusion protein, 【제목키워드】 Production, Impact, fusion, recombinant, SARS-CoV-2 RBD,