Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells

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...

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Bibliographic Details
Published in:Biomolecules (Basel, Switzerland) Switzerland), 2022-08, Vol.12 (9), p.1170
Main Authors: Gerez, Guillaume, Martinez, Jerome, Steinbrugger, Christophe, Bouanich, Sandra, Dimino, Johanna, Piegay, Corine, Combe, Maxime, Berthier, Franck, Daniel, Soizic
Format: Article
Language:English
Subjects:
Cell fusion
chaperone
Coronaviruses
COVID-19
disulphide bond
E484K mutation
Endoplasmic reticulum
Fc receptors
Fusion protein
Gene mutations
Genetic aspects
Mammalian cells
Medical research
Mutation
Plasmids
Production processes
protein disulphide isomerase
Protein folding
Protein research
RBD
Recombinant proteins
SARS-CoV-2
Secretion
Severe acute respiratory syndrome coronavirus 2
Viral proteins
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Summary: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.
ISSN:2218-273X
2218-273X
DOI:10.3390/biom12091170