A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana
Autor: | Martin J. Mueller, Ella Nukarinen, Susanne Berger, Simone Findling, Wolfram Weckwerth, Henrik U. Stotz |
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Rok vydání: | 2014 |
Předmět: |
Tandem affinity purification
biology Arabidopsis Proteins Recombinant Fusion Proteins Arabidopsis Nuclear Proteins Plasma protein binding Plant Science biology.organism_classification Plants Genetically Modified Chromatography Affinity Mass Spectrometry Protein–protein interaction Basic-Leucine Zipper Transcription Factors Biochemistry Calmodulin Seedlings Gene expression Arabidopsis thaliana Signal transduction Transcription factor Research Paper Protein Binding |
Zdroj: | Plant Signaling & Behavior. 9:e29990 |
ISSN: | 1559-2324 1559-2316 |
Popis: | 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. |
Databáze: | OpenAIRE |
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