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Expression and functional validation of Bombyx mori nucleopolyhedrovirus ORF29, a conserved Nudix motif protein

Hui-Qing Chen, Ya-Jing Zhou, Ke-Ping Chen

    Acta Virologica
    |December 4, 2013
    PubMed

    Insights

    The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF29 protein functions as an ADP-ribose pyrophosphatase (ADPRase). This enzyme breaks down ADP-ribose, aiding in understanding viral infection mechanisms.

    Area of Science:

    • Molecular Biology
    • Virology
    • Enzymology

    Background:

    • The Bombyx mori nucleopolyhedrovirus (BmNPV) orf29 gene encodes an early-stage viral protein.
    • BmNPV ORF29 shares homology with the Nudix superfamily, specifically ADP-ribose pyrophosphatase (ADPRase).

    Purpose of the Study:

    • To purify and characterize the recombinant BmNPV ORF29 protein.
    • To investigate the enzymatic activity and properties of BmNPV ORF29.

    Main Methods:

    • Recombinant protein purification using metal chelating affinity chromatography in E. coli.
    • Mass spectroscopic analysis for amino acid sequence confirmation.
    • Enzymatic assays to determine kinetic parameters (Km, Kcat) and optimal activity conditions.

    Main Results:

    • The purified BmNPV ORF29 protein was successfully obtained and verified.
    • The enzyme catalyzes the breakdown of ADP-ribose into AMP and ribose 5-phosphate.
    • Kinetic parameters were determined: Km = 182 μmol/l and Kcat = 5.3 s-1.
    • Optimal enzyme activity was observed at alkaline pH (8.5) with Mg2+ as a cofactor.

    Conclusions:

    • BmNPV ORF29 is an active ADP-ribose pyrophosphatase.
    • Understanding this enzyme's function provides insights into BmNPV replication and pathogenesis.
    • This characterization lays the groundwork for further studies on viral nucleotide metabolism.

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