JP5224491B2 - レセプター結合性物質のスクリーニング方法 - Google Patents
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Description
レセプターを発現し、そしてランダム化したアゴニスト候補物質を細胞表層に提示する酵母を得る工程;および
発現した該アゴニスト候補物質が該レセプターに結合している酵母を検出する工程;を含む。
ランダム化した哺乳類cDNAライブラリー由来の細胞内レセプター共役タンパク質候補物質を細胞内に発現し、アゴニスト結合部位を細胞表層に提示しかつ細胞内レセプター共役タンパク質と相互作用する部位を細胞内に提示するレセプターを細胞膜に発現し、そして、アゴニストを細胞表層に提示する酵母を得る工程;および
該アゴニストと該レセプターとの結合によって該レセプターと該細胞内レセプター共役タンパク質との相互作用が生じた酵母を検出する工程;を含む。
アゴニスト結合部位を細胞表層に提示しかつ細胞内レセプター共役タンパク質と相互作用する部位を細胞内に提示するレセプターを発現し;そして
アゴニストを細胞表層に提示する;酵母を提供する。
該アゴニストと該レセプターとの結合によって該レセプターと該細胞内レセプター共役タンパク質との相互作用が生じた酵母を検出する工程、を含む。
(1−1:欠損酵母株の構築)
酵母S. cerevisiae BY4741株(MATa ura3 leu2 his3 met15)をATCCより入手した。BAR1(プロテアーゼ遺伝子)については、LEU2マーカーの両端がゲノム相同領域となるようにPCRを行った後、酢酸リチウムPCRによる1step遺伝子破壊株作製法(蛋白質核酸酵素,46巻,276頁,2001年)によってノックアウトした。また、FAR1(シグナル伝達により細胞周期阻止を引き起こす遺伝子)については、マーカーとしてカナマイシン耐性遺伝子を用いる酢酸リチウムPCRによる1step遺伝子破壊株作製法(ResGenから購入)によってノックアウトした。次いで、FUS1(シグナル伝達により転写が開始される遺伝子)のC末端にEGFPをコードするDNAを融合した遺伝子を、マーカーとしてHIS3を用いるPCRによる2step遺伝子破壊法によりBY4741株に組込み、欠損酵母BY4741株(MATa ura3 leu2 his3 met15 Δbar1::LEU2 Δfar1::kanMX4 Δfus1::FOS1-EGFP-HIS3)を得た。
本実施例に使用したプラスミドを、表1に列挙する。
上記1−1で得た欠損酵母BY4741株に、上記1−2のようにして得た各プラスミドを、酢酸リチウム法によって導入し、α−因子を表層に提示する酵母を得た。この酵母をウラシルを含まないSRCA培地(YNB(Becton Dickinson and Company製)0.67%、ラフィノース5水和物(シグマ製)2%、カザミノ酸(BD製)2%)100mL中でOD600=1.0まで培養した。さらに、2%ガラクトースを加えて24時間培養した後にサンプリングし、FACSにてソーティングし、FL1フィルターで蛍光を検出した。なお、陽性コントロールとして、上記1−1で得た欠損酵母株にα−因子を分泌型として発現し得るプラスミド(pUESCα)を導入した実験、および陰性コントロールとして、形質転換していない欠損酵母株をα−因子を添加せずに培養するのみの実験も行った。
Claims (2)
- アゴニストのスクリーニング方法であって、
Gタンパク質共役型レセプターを発現して該レセプターのアゴニスト結合部位が細胞表層に提示され、ランダム化したペプチドアゴニスト候補物質を細胞表層局在タンパク質のGPIアンカーを介してまたは細胞表層局在タンパク質の糖鎖結合タンパク質ドメインを介して細胞表層に提示し、該レセプターが酵母の細胞質内のGタンパク質と相互作用して引き起こすシグナル伝達を検出可能とするようにレポーター遺伝子を含み、そしてBAR1遺伝子およびFAR1遺伝子が破壊されている酵母を得る工程;および
該レポーター遺伝子の発現により、発現した該ペプチドアゴニスト候補物質が該レセプターに結合している酵母を検出する工程を含む、方法。 - 前記ペプチドアゴニスト候補物質が、リンカーをさらに介して細胞表層に提示される、請求項1に記載の方法。
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