The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties

  1. Aneta Tarczewska
  2. Marta Kolonko-Adamska
  3. Mirosław Zarębski
  4. Jurek Dobrucki
  5. Andrzej Ożyhar
  6. Beata Greb-Markiewicz

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Abstract

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.03.442392: (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.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    The purified PCR product was cloned into the pET-SUMO vector digested with NcoI and NotI restriction enzymes.
    pET-SUMO
    suggested: RRID:Addgene_73261)
    The final construct (pET-SUMO/N) was confirmed by DNA sequencing.
    pET-SUMO/N
    suggested: None
    Software and Algorithms
    SentencesResources
    The parameters of the N protein, either fused or not fused to 6×His-SUMO, were calculated using the ProtParam server (Gasteiger et al, 2005).
    ProtParam
    suggested: (ProtParam Tool, RRID:SCR_018087)
    Buffer density (ρ = 1.0121 g/cm3) and viscosity (η = 0.010447 mPa×s) were calculated from the composition of the buffer using SEDNTERP software (Harding, 1992).
    SEDNTERP
    suggested: (Sednterp, RRID:SCR_016253)

    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.
    • 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.

  2. Version published to 10.1101/2021.05.03.442392 on bioRxiv