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Plant Signal Behav. 2014;9(10):e972794. doi: 10.4161/15592316.2014.972794.

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

Author information

1
a Julius-von-Sachs-Institute für Biowissenschaften ; Pharmazeutische Biologie; Universität Würzburg ; Würzburg , Germany.

Abstract

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.

KEYWORDS:

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

PMID:
25482810
PMCID:
PMC4622720
DOI:
10.4161/15592316.2014.972794
[Indexed for MEDLINE]
Free PMC Article

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