Escherichia coli recombinant expression of SARS-CoV-2 protein fragments

  1. Bailey E. McGuire
  2. Julia E. Mela
  3. Vanessa C. Thompson
  4. Logan R. Cucksey
  5. Claire E. Stevens
  6. Ralph L. McWhinnie
  7. Dirk F. H. Winkler
  8. Steven Pelech
  9. Francis E. Nano

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Abstract

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli . Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

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  1. Version published to 10.1186/s12934-022-01753-0
  2. ScreenIT

    SciScore for 10.1101/2021.06.22.449540: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Human Serum Samples: The process of the use of human blood products for the investigation of COVID-19 received an ethical approval by Veritas Independent Review Board Inc. (Saint-Laurent, QC, Canada), IRB Protocol 16567-09:39:354-06-2020.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To detect the bound antibodies, the arrays were washed with T-TBS three times followed by the incubation with goat anti-human IgG+IgA+IgM pAb or donkey-anti-rabbit IgG (HRP conjugates, 1:20,000 dilution; Jackson ImmunoResearch, West Grove, Pennsylvania, USA).
    anti-human IgG+IgA+IgM
    suggested: None
    IgG
    suggested: None
    Recombinant DNA
    SentencesResources
    Since the native CBM9 gene has an internal Esp3I site and pRSET5A has internal BsaI sites, the plasmid and CBM9 amplicons were separately digested with Esp3I and BsaI and then joined by a standard DNA ligation.
    pRSET5A
    suggested: None
    Once initial clones were sequence verified and shown to produce the appropriate protein product, further recombinants were constructed using the pRSET5A::CBM9-id-c clone as the backbone.
    pRSET5A::CBM9-id-c
    suggested: None
    To make a plasmid encoding just the CBM9-(TP)4P (no SARS-CoV-2 fragment) or CBM9-N (containing a nucleocapsid epitope), plasmid pRSET5A::CBM9-id-a was amplified with primers nF2-R5A-CBD and nR-R5A-Flex; or F-nucl-ep and R-nucl-ep and Esp3I digested and ligated with a strategy depicted in Fig. 1C.
    pRSET5A::CBM9-id-a
    suggested: None
    Software and Algorithms
    SentencesResources
    The recombinant clones expressing CBM9-(PT)4P, CBM9-ID-F, CBM9-ID-H1 and CBM9-N have been deposited with AddGene (https://www.addgene.org/).
    https://www.addgene.org/
    suggested: (Addgene, RRID:SCR_002037)

    Results from OddPub: Thank you for sharing your data.

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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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  3. Version published to 10.1101/2021.06.22.449540v1 on bioRxiv