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

J Vis Exp. 2019 May 1;(147):10.3791/59527. doi: 10.3791/59527.

Abstract

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Aspergillus nidulans / enzymology*
  • Chickens
  • Chromatography, Affinity / methods*
  • Enzyme Assays / methods*
  • Histone Deacetylases / genetics
  • Histone Deacetylases / metabolism*
  • Hydroxamic Acids / pharmacology
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Hydroxamic Acids
  • Recombinant Fusion Proteins
  • trichostatin A
  • Histone Deacetylases