JP4883640B2 - 新規糖固定化金属ナノ粒子およびこれを用いて糖−タンパク質相互作用を測定する方法、並びに糖−タンパク質相互作用体からタンパク質を回収する方法 - Google Patents
新規糖固定化金属ナノ粒子およびこれを用いて糖−タンパク質相互作用を測定する方法、並びに糖−タンパク質相互作用体からタンパク質を回収する方法 Download PDFInfo
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Description
M.S.Stoll et al.,Eur.J.Biochem.267,1795−1804,2000 M.Schwarz et al.,Glycobiol.13,no.11,749−754,2003 H.Otsuka et al.,J.Am.Chem.Soc.123,8226−8230,2001 J.M.de la Fuente et al.,Angew.Chem.Int.Ed.40,No.12,2258−2260,2001 しかしながら、標識した糖鎖やタンパク質を用いて糖−タンパク質相互作用を測定する方法は、標識という前処理が必要となるため、標識効果が測定のばらつきに大きく影響するという問題がある。一方、非標識の方法であっても、従来のSPRやQCMでは非常に高価な装置が必要であり、簡便な測定は困難であった。
本明細書において、「糖固定化金属ナノ粒子」とは、以下に詳述するリガンド複合体と、任意の金属とを結合させてなるものである。
本発明に係る糖−タンパク質相互作用の測定方法は、糖固定化金属ナノ粒子を含む溶液と、上記糖固定化金属ナノ粒子の末端に位置する糖を認識するタンパク質とを混和することによって、糖とタンパク質とを相互作用させ、糖−タンパク質相互作用体を生成させる工程を含むものである。
一実施形態において、本発明に係る糖−タンパク質相互作用体からタンパク質を回収する方法は、糖固定化金属ナノ粒子を含む溶液と、上記糖固定化金属ナノ粒子の末端に位置する糖を認識するタンパク質とを混和して糖とタンパク質とを相互作用させ、糖−タンパク質相互作用体を生成させる工程と、当該糖−タンパク質相互作用体と水とを混和した混和液のpHを5以下とする工程と、を含んでいる。
テトラクロロ金(III)酸ナトリウムの終濃度が1mM、水素化ホウ素ナトリウムの終濃度が5mMとなるように、テトラクロロ金(III)酸ナトリウムと水素化ホウ素ナトリウムとを水を用いて激しく攪拌・混和し、金ナノ粒子溶液を調製した。リガンド複合体としては、チオクト酸、m−フェニレンジアミンおよびマルトースからなる化合物(質量比 1:1:1)を用い、終濃度が100μMになるように適宜水で調製した上記リガンド複合体を、上記金ナノ粒子溶液に添加して激しく攪拌・混和し、粗マルトース固定化金ナノ粒子のコロイド溶液を調製した。
96穴タイタープレートに、マルトースを認識するタンパク質であるコンカナバリンA(以下「ConA」と記載する;EYラボラトリーズ製)の希釈系列およびマルトースを認識しないタンパク質であるウシ血清アルブミン(以下「BSA」と記載する;SIGMA製)の希釈系列(PBS−T(0.05%))を10μMから作製(25μl)し、そこへ上記マルトース固定化金ナノ粒子のコロイド溶液を1:1で添加した。30分から2時間放置し、マルトース固定化金ナノ粒子が完全に凝集し終わった後、目視および紫外可視吸収スペクトル(540nm)の測定により結果を評価した。
エッペンドルフチューブに250μg/ml(PBS−T(0.05%))のConAを100μl分注し、実施例1で調製したマルトース固定化金ナノ粒子のコロイド溶液を100μl添加してボルテックスで10秒以上撹拌した。約2時間撹拌放置した後、遠心分離にて糖−タンパク質相互作用体を沈殿させ、上清を回収した後、当該糖−タンパク質相互作用体をPBS−T 0.05%、水で数回洗浄した。当該洗浄後、200mMのグルコース水溶液を100μl添加し約1時間放置した。結果は、糖−タンパク質相互作用体からConAが解離したことにより、ConAが糖−タンパク質相互作用体からマルトース固定化金ナノ粒子のコロイド溶液へ戻ることを目視することおよび紫外可視吸光スペクトルを測定することにより評価した。
本実施例では、マルトース、ラクトース、キトビオースをそれぞれ固定して、糖固定化金ナノ粒子のコロイド溶液を調製した。そして、糖固定化金ナノ粒子のコロイド溶液について、レクチンと特異的に結合するかどうかを調べることにより、糖−タンパク質相互作用を測定した。
本実施例は、糖−タンパク質相互作用体として、実施例3に記載した以外のものを用いた場合においてもタンパク質を回収できることを示すものである。
本実施例では、SDS−PAGEを用いて、糖固定化金ナノ粒子のコロイド溶液が保持可能なタンパク質の量を検定することを目的として、以下の実験を行った。
既に説明したように、タンパク質と糖固定化金属ナノ粒子とを反応させる際に予めタンパク質が認識可能な様々な糖鎖を添加しておくことにより糖−タンパク質相互作用に起因する凝集を阻害することができれば、より詳しくタンパク質の機能を解析することができる。例えば、タンパク質に対してどの糖鎖がより強固に結合するかということ等の機能解析が可能になると考えられる。
Claims (5)
- 末端に芳香族アミノ基を有するとともに、主鎖に炭素−窒素結合を有していてもよい炭化水素誘導鎖を備えるとともに、硫黄原子を含む炭化水素構造を備えているリンカー化合物と、還元末端を有する糖とが上記アミノ基を介して結合している構造を有するリガンド複合体と、金とを結合させてなる糖固定化金属ナノ粒子であって、
テトラクロロ金(III)酸ナトリウムと、水素化ホウ素ナトリウムと、を混和することによって調製された金ナノ粒子の溶液に、上記構造を有するリガンド複合体を混和することによって調製され、
安定であり、
上記金ナノ粒子の溶液と、上記リガンド複合体とを混和することによって調製された糖固定化金属ナノ粒子の溶液に対し、テトラクロロ金(III)酸ナトリウムが0.5mM〜4mM、水素化ホウ素ナトリウムが金イオンのモル濃度の3〜10倍モル濃度、上記リガンド複合体が10μM〜1000μM含まれており、
上記糖固定化金属ナノ粒子は、糖をタンパク質と相互作用させることによって糖−タンパク質相互作用体を生成しうることを特徴とする糖固定化金属ナノ粒子。 - 上記硫黄原子を含む炭化水素構造は、S−S結合またはSH基を含む炭化水素構造であることを特徴とする請求項1に記載の糖固定化金属ナノ粒子。
- 請求項1または2に記載の糖固定化金属ナノ粒子を含む溶液と、上記糖固定化金属ナノ粒子の末端に位置する糖を認識するタンパク質とを混和することによって、糖とタンパク質とを相互作用させ、糖−タンパク質相互作用体を生成させる工程を含むことを特徴とする糖−タンパク質相互作用の測定方法。
- 請求項1または2に記載の糖固定化金属ナノ粒子を含む溶液と、上記糖固定化金属ナノ粒子の末端に位置する糖を認識するタンパク質とを混和して糖とタンパク質とを相互作用させ、糖−タンパク質相互作用体を生成させる工程と、
当該糖−タンパク質相互作用体と水とを混和した混和液のpHを5以下とする工程と、を含むことを特徴とする糖−タンパク質相互作用体からタンパク質を回収する方法。 - 請求項1または2に記載の糖固定化金属ナノ粒子を含む溶液と、上記糖固定化金属ナノ粒子の末端に位置する糖を認識するタンパク質とを混和して、糖とタンパク質とを相互作用させ、糖−タンパク質相互作用体を生成させる工程と、
当該糖−タンパク質相互作用体と上記タンパク質が認識可能な糖とを混和する工程と、
を含むことを特徴とする糖−タンパク質相互作用体からタンパク質を回収する方法。
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