薬剤をリガンドとして固定化したFG beadsを用いて、標的タンパク質を精製する手法である「ケミカルプルダウン法」を利用して、生体内分子の機能や反応のメカニズムを分子レベルから解析します。FG beadsは、様々な種類の化合物が固定化でき、非特異的な吸着が極めて少ないため、ケミカルバイオロジーを最も得意としています。
化合物の「1」および「2」が活性に寄与しており、FG beadsへの固定化で失活するため、「3」の部位での固定化が必要になります。
FG beads上に化合物(薬剤)を固定化した後に、標的タンパク質を回収(結合)する。
化合物(薬剤)と標的タンパク質を結合させた後に、FGbeadsにて「化合物+標的タンパク質」の複合体を回収する。
アジド、アルキン、ビオチン、tagなどの導入が必要。
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固定化の 難易度 |
固定化部位の官能基(化合物側) | 使用ビーズ | |
---|---|---|---|
低 | 直接法 | NH2 | NHS beadsCOOH beads |
COOH | NH2 beadsOH beads | ||
Azide | Alkyne beads | ||
Alkyne (Ethynyl) | Azide beads | ||
OH(フェノール性) | Epoxy beads | ||
OH | COOH beads | ||
間接法 | Azide | Alkyne beads | |
高 | Alkyne (Ethynyl) | Azide beads |
化合物の固定化量は多くても少なくてもダメ。まずは、仕込み量を4点振って各化合物に最適な固定化条件を見つけることが大切だよ。 |
競合阻害やドラッグエリューションを行うことで結合タンパク質のバンドの絞り込みや、結合タンパク質の特異性評価を行うことも可能です。
薬剤標的タンパク質の精製 ‐Methotrexate
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薬剤標的タンパク質の精製 ‐ビオチン化MTX
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キナーゼ阻害剤Bisindolylmaleimide Ⅹの結合タンパク質の精製
国内製薬企業との実施
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キナーゼ阻害剤Bisindolylmaleimide VIIIの標的タンパク質の探索~ビーズ上で精製されたタンパク質の分析
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