Designed SARS-CoV-2 receptor binding domain variants form stable monomers.
COVID-19
antibody assay validation
antigen stability
receptor binding domain
recombinant expression
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
May 2022
May 2022
Historique:
revised:
08
01
2022
received:
08
08
2021
accepted:
10
01
2022
pubmed:
26
1
2022
medline:
11
5
2022
entrez:
25
1
2022
Statut:
ppublish
Résumé
The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence. The authors found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates.
Identifiants
pubmed: 35078277
doi: 10.1002/biot.202100422
pmc: PMC9011732
doi:
Substances chimiques
Antibodies, Neutralizing
0
Antibodies, Viral
0
Spike Glycoprotein, Coronavirus
0
spike protein, SARS-CoV-2
0
Cysteine
K848JZ4886
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2100422Subventions
Organisme : Bundesministerium fuer Digitalisierung und Wirtschaftsstandort
Organisme : Universitaet fuer Bodenkultur Wien
Organisme : Equipment Boku Vienna Institute Of Biotechnology
Organisme : Vienna Science and Technology Fund
ID : COV20-016
Organisme : Austrian Science Fund FWF
ID : W 1224
Pays : Austria
Organisme : Oesterreichische Nationalstiftung fuer Forschung, Technologie und Entwicklung
Organisme : Christian Doppler Forschungsgesellschaft
Organisme : Austrian Science Fund
ID : W1224-B09
Informations de copyright
© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
Références
Cell Discov. 2021 Sep 7;7(1):82
pubmed: 34493710
Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W382-8
pubmed: 15980494
Cell. 2020 Apr 16;181(2):281-292.e6
pubmed: 32155444
Nature. 2020 Oct;586(7830):572-577
pubmed: 32726802
Curr Protoc Microbiol. 2020 Jun;57(1):e100
pubmed: 32302069
Bioinformatics. 2011 Jun 15;27(12):1711-2
pubmed: 21505037
Vaccines (Basel). 2020 Nov 04;8(4):
pubmed: 33158147
Nature. 2020 Aug;584(7821):443-449
pubmed: 32668443
Appl Microbiol Biotechnol. 2021 May;105(10):4153-4165
pubmed: 33959781
Glycobiology. 2020 Dec 9;30(12):981-988
pubmed: 32363391
Cell. 2020 Sep 3;182(5):1284-1294.e9
pubmed: 32730807
Biotechnol Bioeng. 2021 Jun;118(6):2202-2219
pubmed: 33624859
Nature. 2020 May;581(7807):215-220
pubmed: 32225176
Nature. 2020 Mar;579(7798):265-269
pubmed: 32015508
PLoS One. 2021 Feb 2;16(2):e0242890
pubmed: 33529230
Anal Chem. 2021 Nov 2;93(43):14392-14400
pubmed: 34670086
Biochim Biophys Acta Gen Subj. 2021 Jun;1865(6):129893
pubmed: 33731300
Biotechnol J. 2022 May;17(5):e2100422
pubmed: 35078277
Glycobiology. 2021 Apr 1;31(3):181-187
pubmed: 32886791
Sci Rep. 2020 Dec 11;10(1):21779
pubmed: 33311634
Front Immunol. 2021 May 24;12:641447
pubmed: 34108961
Nat Med. 2020 Jul;26(7):1033-1036
pubmed: 32398876
J Chem Theory Comput. 2021 Apr 13;17(4):2479-2487
pubmed: 33689337
Cell. 2020 Aug 6;182(3):722-733.e11
pubmed: 32645327
Science. 2020 May 8;368(6491):630-633
pubmed: 32245784
J Virol. 2020 Mar 17;94(7):
pubmed: 31996437
Viruses. 2021 Mar 25;13(4):
pubmed: 33806155
Cell. 2020 Sep 3;182(5):1295-1310.e20
pubmed: 32841599
Anal Chem. 2021 Feb 2;93(4):2003-2009
pubmed: 33406838
Protein Expr Purif. 2021 Jul;183:105861
pubmed: 33667651
Biotechnol J. 2021 Jun;16(6):e2000566
pubmed: 33481336
J Med Virol. 2021 Feb;93(2):924-933
pubmed: 32706425
Science. 2020 Mar 27;367(6485):1444-1448
pubmed: 32132184
Sci Rep. 2020 Oct 19;10(1):17698
pubmed: 33077899
Science. 2020 Jul 17;369(6501):330-333
pubmed: 32366695
EBioMedicine. 2021 May;67:103348
pubmed: 33906067
Cell Host Microbe. 2020 Oct 7;28(4):586-601.e6
pubmed: 32841605
ACS Cent Sci. 2020 Oct 28;6(10):1722-1734
pubmed: 33140034
Front Plant Sci. 2021 Jun 15;12:689104
pubmed: 34211491
Cell. 2020 Apr 16;181(2):271-280.e8
pubmed: 32142651
Mol Biol. 2019;53(3):323-334
pubmed: 32214478