Production of novel Spike truncations in Chinese hamster ovary cells

  1. Shiaki A. Minami
  2. Seongwon Jung
  3. Yihan Huang
  4. Bradley S. Harris
  5. Matthew W. Kenaston
  6. Roland Faller
  7. Somen Nandi
  8. Karen A. McDonald
  9. Priya S. Shah

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Abstract

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, Spike receptor binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. Next, we develop 8 Spike truncations (T1-T8) in pursuit of a truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest T1 is a promising Spike alternative for use in various applications.

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  1. ScreenIT

    SciScore for 10.1101/2021.12.06.471489: (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
    Membranes were stained overnight at 4°C with a 1:1000 diluted mouse anti-his primary antibody (MCA1396, RRID:AB_322084, Bio-Rad) and then for 1 hour at room temperature with a 1:4000 diluted rabbit anti-mouse HRP secondary antibody (SouthernBiotech Cat# 6170-05, RRID:AB_2796243, Birmingham, AL).
    with a mouse anti-his
    detected: (Bio-Rad Cat# MCA1396, RRID:AB_322084)
    anti-mouse HRP
    detected: (SouthernBiotech Cat# 6170-05, RRID:AB_2796243)
    Membranes were blocked overnight in 1% casein, stained with 1:1000 diluted mouse anti-his primary antibody and stained with 1:4000 diluted rabbit anti-mouse secondary antibody.
    anti-his
    suggested: None
    anti-mouse
    suggested: None
    Plates were incubated with 1:1000 rabbit anti-RBD primary antibody (Sino Biological Cat# 40592-R001, RRID:AB_2857936, Wayne, PA), then 1:6000 or 1:4000 goat anti-rabbit, HRP secondary antibody (SouthernBiotech Cat# 4030-05, RRID:AB_2687483) in PBS with 1% BSA for purified or crude proteins, respectively.
    anti-RBD
    suggested: None
    with rabbit
    detected: (Sino Biological Cat# 40592-R001, RRID:AB_2857936)
    anti-rabbit
    detected: (SouthernBiotech Cat# 4030-05, RRID:AB_2687483)
    Indirect ELISAs were performed to assess the sensitivities of CHO-expressed proteins to a human anti-Spike monoclonal antibody CR3022 (NR-52392, BEI Resources, RRID:AB_2848080) and a rabbit anti-Spike polyclonal antibody (PAb, eEnzyme, SCV2-S-100, RRID:AB_2893135, Gaithersburg, MD).
    anti-Spike
    detected: (eEnzyme Cat# SCV2-S-100, RRID:AB_2893135)
    proteins a
    detected: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    Plates were loaded with 100 µL/well goat anti-hIgG, HRP secondary antibody at 1:4000 in PBS containing 1% BSA.
    anti-hIgG
    suggested: (Antibodies-Online Cat# ABIN288163, RRID:AB_10795674)
    For the PAb, 3-fold serial dilutions starting at 400 ng/well of rabbit anti-Spike primary antibody were used (PAb, SCV2-S-100, eEnzyme), and a 1:4000 goat anti-rabbit IgG, HRP secondary antibody was used instead.
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Standard curves for quantifying Spike and RBD were generated using serial dilutions of Sf9 insect Spike (NR-52308, BEI Resources, Manassas, VA) and HEK293F human RBD (NR-52366, BEI Resources), respectively
    HEK293F
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: pCAGGS-Spike and pCAGGS-RBD were gifted from Florian Krammer (Amanat et al., 2020).
    pCAGGS-Spike
    suggested: None
    Truncations T1-T8 were synthesized (GenScript, Piscataway, NJ) with overhangs for insertion into pCAGGS vectors (Table S1).
    pCAGGS
    suggested: RRID:Addgene_127347)
    Truncations were inserted into pCAGGS vectors via Gibson Assembly of pCAGGS-RBD digested with XbaI and XhoI.
    pCAGGS-RBD
    suggested: None
    Software and Algorithms
    SentencesResources
    Possible structural and binding motifs for the truncations were determined with PredictProtein (Yachdav et al., 2014).
    PredictProtein
    suggested: None
    A standard curve was generated via densitometry through ImageJ, and primary band intensities for the samples were interpolated to quantify concentrations.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Amber ff14SB and Glycam06 forcefields (Kirschner et al., 2008; Maier et al., 2015) were used and generated using acpype.py following the method shown previously (Bernardi et al., 2017, 2019).
    Amber
    suggested: (AMBER, RRID:SCR_016151)
    Simulations were conducted using the Gromacs 2019.1 suite with similar energy minimization procedure as in previous simulations (Abraham et al., 2015; Pronk et al., 2013; Van Der Spoel et al., 2005) including ones involving glycosylated RBD (Bernardi et al., 2017; Huang et al., 2021).
    Gromacs
    suggested: (GROMACS, RRID:SCR_014565)

    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 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.

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  2. Version published to 10.1101/2021.12.06.471489v1 on bioRxiv