The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression

  1. Mariano Maffei
  2. Linda Celeste Montemiglio
  3. Grazia Vitagliano
  4. Luigi Fedele
  5. Shaila Sellathurai
  6. Federica Bucci
  7. Mirco Compagnone
  8. Valerio Chiarini
  9. Cécile Exertier
  10. Alessia Muzi
  11. Giuseppe Roscilli
  12. Beatrice Vallone
  13. Emanuele Marra

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Abstract

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

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  1. Version published to 10.3390/biom11121812
  2. ScreenIT

    SciScore for 10.1101/2021.09.17.460782: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Detection was achieved using horseradish peroxidase-conjugate secondary antibody anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515) and visualized with ECL (Cytiva RPN2232).
    anti-mouse
    suggested: (Bio-Rad Cat# 170-6516, RRID:AB_11125547)
    Antibodies: The primary antibodies used in this study are: rabbit anti-SARS-CoV-2 Spike S1 Subunit (Sino Biological, 40150-T62)
    anti-SARS-CoV-2
    suggested: None
    Secondary antibody used are: horseradish peroxidase-conjugate anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515).
    anti-rabbit
    suggested: (Bio-Rad Cat# 170-6515, RRID:AB_11125142)
    After washing, AP-conjugated goat anti-rat IgG antibody (SIGMA A8438) or AP-conjugated goat anti-rabbit IgG antibody (SIGMA A8025) was added, and the plates were further incubated for 1 hour at RT.
    anti-rat IgG
    suggested: (Sigma-Aldrich Cat# A8438, RRID:AB_258391)
    anti-rabbit IgG
    suggested: (Sigma-Aldrich Cat# A8025, RRID:AB_258372)
    FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)
    anti-RBD
    suggested: None
    anti-human IgG
    suggested: (Molecular Probes Cat# A-11013, RRID:AB_141360)
    Experimental Models: Cell Lines
    SentencesResources
    Hi-5 cells (BTI-TN-5B1-4) (Gibco #B85502) were cultured in Express Five™ SFM (Serum-Free Media) medium (Gibco #B85502 Expression Systems) at a cell density of 0.5 x 106 cells/mL and infected with recombinant virus.
    Hi-5
    suggested: None
    FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)
    Vero E6
    suggested: None
    After a wash step in 1× kinetic buffer for 120 s, the ACE2-Fc-captured biosensor tips were then submerged for 300 s in wells containing different concentrations of antigen (RBD E. coli, insect, and HEK-293) to evaluate association curves, followed by 900 s of dissociation time in kinetic buffer.
    HEK-293
    suggested: None
    Recombinant DNA
    SentencesResources
    RBD protein production in E. coli: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned with a C-terminal 6x-His tag into a pET-21a(+) plasmid. E. coli BL21 Star™ (DE3) (genotype: F−ompT hsdSB (rB−, mB−) galdcmrne131) competent cells, and E. coli Lemo21 (DE3) (genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS/ pLemo(CamR)) competent cells were transformed with 100 ng of plasmid of interest.
    pET-21a(+
    suggested: RRID:Addgene_12669)
    RBD protein production in insect cells: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned into a pFAST-bac1 plasmid downstream of the gp64 signal sequence to promote secretion, along with a C-terminal 8x-His tag for affinity purification.
    pFAST-bac1
    suggested: None
    Software and Algorithms
    SentencesResources
    Raw data were analyzed using the Biopharma Finder 2.1 software from ThermoFisher Scientific.
    Biopharma Finder
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, the high costs (resources), the time-consuming production, the requirement of specific equipment, and access to dedicated facilities could be a limitation for many laboratories or for the industrial production. By contrast, the bacterial-derived RBD offers a low production cost, a broader availability, and easy handling as main advantages, which make it more accessible. However, limitations in the quality of the produced sample include the absence of glycosylation that partially affects protein stability and efficiency, the presence of heterogeneous folded populations, and the relative low production yields which may result in a final product not eligible for some clinical and medical applications. Overall, all the recombinantly produced RBDs represent valuable tools for research purposes against the pandemic. Recently, expression and purification strategies described in this article have been also proved to be successful in the production of mutants of RBD corresponding to the variants of concern.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.09.17.460782v1 on bioRxiv