Isolation of transcription factor complexes from **Arabidopsis** cell suspension cultures by tandem affinity purification
Faculty of Sciences. Biology
Berlin :Springer, 2011
Plant transcription factors / Yuan, L. [edit.]; et al. [edit.]
University of Antwerp
Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the proteinprotein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study proteinprotein interactions under nearphysiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a treptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GSbased TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.