title: Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.
abstract: 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 protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological 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 streptavidin-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 GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.
authors: Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Gent, Belgium. jelee@psb.vib-ugent.be
authors: Jelle
authors: J
authors: Van Leene
authors: Dominique
authors: D
authors: Eeckhout
authors: Geert
authors: G
authors: Persiau
authors: Eveline
authors: E
authors: Van De Slijke
authors: Jan
authors: J
authors: Geerinck
authors: Gert
authors: G
authors: Van Isterdael
authors: Erwin
authors: E
authors: Witters
authors: Geert
authors: G
authors: De Jaeger
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D046912:Multiprotein Complexes / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D012097:Repressor Proteins / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D046912:Multiprotein Complexes; D012097:Repressor Proteins; D014157:Transcription Factors; C112279:COI1 protein, Arabidopsis; C554741:JAZ1 protein, Arabidopsis; C473611:MYC2 protein, Arabidopsis; C549252:NINJA protein, Arabidopsis; C511291:At1g15750 protein, Arabidopsis