Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Gerez, Guillaume, Martinez, Jerome, Steinbrugger, Christophe, Bouanich, Sandra, Dimino, Johanna, Piegay, Corine, Combe, Maxime, Berthier, Franck, Daniel, Soizic
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496381/
https://www.ncbi.nlm.nih.gov/pubmed/36139010
http://dx.doi.org/10.3390/biom12091170
_version_ 1784794254831255552
author Gerez, Guillaume
Martinez, Jerome
Steinbrugger, Christophe
Bouanich, Sandra
Dimino, Johanna
Piegay, Corine
Combe, Maxime
Berthier, Franck
Daniel, Soizic
author_facet Gerez, Guillaume
Martinez, Jerome
Steinbrugger, Christophe
Bouanich, Sandra
Dimino, Johanna
Piegay, Corine
Combe, Maxime
Berthier, Franck
Daniel, Soizic
author_sort Gerez, Guillaume
collection PubMed
description 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.
format Online
Article
Text
id pubmed-9496381
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-94963812022-09-23 Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells Gerez, Guillaume Martinez, Jerome Steinbrugger, Christophe Bouanich, Sandra Dimino, Johanna Piegay, Corine Combe, Maxime Berthier, Franck Daniel, Soizic Biomolecules Article 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. MDPI 2022-08-24 /pmc/articles/PMC9496381/ /pubmed/36139010 http://dx.doi.org/10.3390/biom12091170 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gerez, Guillaume
Martinez, Jerome
Steinbrugger, Christophe
Bouanich, Sandra
Dimino, Johanna
Piegay, Corine
Combe, Maxime
Berthier, Franck
Daniel, Soizic
Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title_full Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title_fullStr Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title_full_unstemmed Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title_short Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
title_sort impact of sars-cov-2 rbd mutations on the production of a recombinant rbd fusion protein in mammalian cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496381/
https://www.ncbi.nlm.nih.gov/pubmed/36139010
http://dx.doi.org/10.3390/biom12091170
work_keys_str_mv AT gerezguillaume impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT martinezjerome impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT steinbruggerchristophe impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT bouanichsandra impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT diminojohanna impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT piegaycorine impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT combemaxime impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT berthierfranck impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells
AT danielsoizic impactofsarscov2rbdmutationsontheproductionofarecombinantrbdfusionproteininmammaliancells