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    Biotechnology journal. 2022 Jan 25. doi: 10.1002/biot.202100422
    Designed SARS-CoV-2 receptor binding domain variants form stable monomers.
    Klausberger M1,  Kienzl NF2,  Stadlmayr G3,  Grünwald-Gruber C4,  Laurent E5,  Stadlbauer K6,  Stracke F7,  Vierlinger K8,  Hofner M9,  Manhart G10,  Gerner W11,  Grebien F12,  Weinhäusel A13,  Mach L14,  Wozniak-Knopp G15
    Author information
    1Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    2Institute of Plant Biotechnology and Cell Biology, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    3Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    4Institute of Biochemistry, Department of Chemistry and BOKU Core Facility Mass Spectrometry, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    5Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    6Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    7Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    8Competence Unit Molecular Diagnostics, Center for Health and Bioresources, Austrian Institute of Technology, Giefinggasse 4, Vienna, 1210, Austria.
    9Competence Unit Molecular Diagnostics, Center for Health and Bioresources, Austrian Institute of Technology, Giefinggasse 4, Vienna, 1210, Austria.
    10Institute of Medical Biochemistry, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, Austria.
    11Institute of Immunology, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, Austria.
    12Institute of Medical Biochemistry, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, Austria.
    13Competence Unit Molecular Diagnostics, Center for Health and Bioresources, Austrian Institute of Technology, Giefinggasse 4, Vienna, 1210, Austria.
    14Institute of Plant Biotechnology and Cell Biology, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    15Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, Vienna, 1190, Austria.
    Abstract

    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. We 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. We found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, we show 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. This article is protected by copyright. All rights reserved.


    This article is protected by copyright. All rights reserved.

    KEYWORDS: COVID-19, antibody assay validation, antigen stability, receptor binding domain, recombinant expression

    Publikations ID: 35078277
    Quelle: öffnen
     
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