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    Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay


    Bauer, Ingo and Pidroni, Angelo and Bayram, Ozgur and Brosch, Gerald and Graessle, Stefan (2019) Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay. Journal of Visualized Experiments, 147 (e59527). ISSN 1940-087X

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    Abstract

    Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genomemining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exerttheir function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenouscomplexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, islaborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thusturned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of activeRpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then beused for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay canbe completed within two days. The video component of this article can be found at https://www.jove.com/video/59527/

    Item Type: Article
    Additional Information: Citation: Bauer, I., Pidroni, A., Bayram, Ö., Brosch, G., Graessle, S. Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay. J. Vis. Exp. (147), e59527, doi:10.3791/59527 (2019). Copyright © 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 UnportedLicense
    Keywords: Biochemistry; Issue 147; Histone Deacetylase; Chromatin; Enzyme Activity; HDAC Assay; Affinity Purification; Chromatography; Tobacco Etch Virus Protease; Aspergillus nidulans; Filamentous Fungi; Single-step Protein Enrichment; Multiprotein Complexes;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 10996
    Identification Number: https://doi.org/10.3791/59527
    Depositing User: Ozgur Bayram
    Date Deposited: 28 Aug 2019 16:13
    Journal or Publication Title: Journal of Visualized Experiments
    Publisher: JOVE
    Refereed: Yes
    URI:

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