オーキシン受容体のクローニングに向けた酵母three-hybrid法の改良

概要

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Our objective is cloning of an auxin receptor cDNA with a yeast three-hybrid system which can detect small ligand-protein interaction in yeast cells and enables us to isolate a cDNA encoding a ligand-binding protein directly. In the original system, the interaction between a rat glucocorticoid receptor (GR) and dexamethasone (Dex) has been used as a hook. To improve the sensitivity of the system, we applied the combination of His-tag and Ni-NTA which interacts stronger than that of GR and Dex. In the hook plasmid 6 × His-tag was fused at the C-terminus of GAL4 DNA binding domain, and on the other hand, Dex binding domain for rat GR was fused to the transcriptional activation domain of GAL4 in a fish construct. As a bait ligand, the NTA was covalently crosslinked to Dex, and the carboxyl group of NTA was also protected as acetoxymethylester, which confers higher membrane permeability and is easily hydrolyzed in yeast cells. Yeast cells (strain Ah109) were transfomed with the two plasmids and were plated on medium containing Ni^<2+> and the synthesized bait ligands.
植物化学調節学会の論文
2000-11-02