Identification of protein complexes from filamentous fungi with tandem affinity purification

Methods Mol Biol. 2012:944:191-205. doi: 10.1007/978-1-62703-122-6_14.

Abstract

Fungal molecular biology has benefited from the enormous advances in understanding protein-protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extracts under mild conditions. We codon-optimized tags and established TAP, previously not applicable to filamentous fungi, for the model organism Aspergillus nidulans. We could identify by this method the trimeric Velvet complex VelB/VeA/LaeA or the eight subunit COP9 signalosome. Here, we describe an optimized protocol for A. nidulans which can also be adapted to other filamentous fungi.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus nidulans / chemistry*
  • Benzenesulfonates / chemistry
  • Chromatography, Affinity / methods*
  • Chromatography, High Pressure Liquid
  • Databases, Protein
  • Electrophoresis, Polyacrylamide Gel
  • Fungal Proteins / chemistry*
  • Fungal Proteins / isolation & purification*
  • Fungal Proteins / metabolism
  • Mass Spectrometry
  • Proteolysis
  • Silver Staining
  • Trypsin / metabolism

Substances

  • Benzenesulfonates
  • Fungal Proteins
  • Trypsin
  • brilliant blue