A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.
Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.
['Stotz|Henrik U|HU|', 'Findling|Simone|S|', 'Nukarinen|Ella|E|', 'Weckwerth|Wolfram|W|', 'Mueller|Martin J|MJ|', 'Berger|Susanne|S|']
D017360:Arabidopsis D029681:Arabidopsis Proteins D050976:Basic-Leucine Zipper Transcription Factors D002147:Calmodulin D002846:Chromatography, Affinity D013058:Mass Spectrometry D009687:Nuclear Proteins D030821:Plants, Genetically Modified D011485:Protein Binding D011993:Recombinant Fusion Proteins D036226:Seedlings
12-oxo-phytodienoic acid CBB, calmodulin binding buffer CBP, calmodulin-binding peptide CaMV, cauliflower mosaic virus FDR, false discovery rate MS, mass spectrometry OPDA, 12-oxo-phytodienoic acid PGA1, prostaglandin A1 PPA1, phytoprostane A1 RubisCo, ribulose-1,5-bisphosphate carboxylase SA, salicylic acid SAR, systemic acquired resistance TAP, tandem affinity purification TEV, tobacco etch virus Y2H, yeast 2-hybrid bZIP, basic region/leucine zipper motif glutathione-S-transferase lipid stress protein complex thale cress