Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

  1. Matthew Stuible
  2. Christian Gervais
  3. Simon Lord-Dufour
  4. Sylvie Perret
  5. Denis L’Abbe
  6. Joseph Schrag
  7. Gilles St-Laurent
  8. Yves Durocher

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Abstract

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHO BRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.08.286732: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Protein-coding DNA sequences were codon-optimized for CHO cells, synthesized and cloned into the pTT5™ vector [18] by Genscript.
    CHO
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    2.2 Expression platforms: Transfection and post-transfection culture of CHO-3E7 and 293-6E cells in FreeStyle F17 media were performed as described previously [19, 20].
    293-6E
    suggested: RRID:CVCL_HF20)
    The method for transient expression in CHOBRI/rcTA cells [21] is based on a published method for high-density transfection of CHO-3E7 cells [19], with several modifications.
    CHOBRI/rcTA
    suggested: None
    CHO-3E7
    suggested: RRID:CVCL_JY74)
    Software and Algorithms
    SentencesResources
    Weighted average molecular mass (MMALS) was calculated in ASTRA 6.1 software (Wyatt) using a protein concentration determined from the refractive index signal with a dn/dc value of 0.185.
    ASTRA
    suggested: (ASTRA, RRID:SCR_016255)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    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.

  2. Version published to 10.1101/2020.09.08.286732 on bioRxiv