Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein–protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni2+ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages97-108
Number of pages12
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2356
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Filamentous fungi
  • Low-abundance protein complexes
  • Magnaporthe oryzae
  • Mass spectrometry
  • Tandem affinity purification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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