ディスプレイクローニングと計算科学による結合タンパク質の戦略的決定

• Project/Area Number: 15651101
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Living organism molecular science
• Research Institution: Tokyo University of Science
• Principal Investigator: 菅原 二三男 東京理科大学, 理工学部, 教授 (30192123)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: ジベレリン / ジベレリン結合タンパク / ファージディスプレイ法 / Ipha-exansin2 / nodulin / イネ(Orza sativa) / 培養細胞ライブラリ / 出芽組織ライブラリ / ZD1839 / イレッサ / ファージディスプレイ / カンプトテシン / IGF-1R / EGFR

Research Abstract

植物ホルモン,ジベレリンの結合タンパク質を決定するため,イネcDNAをファージに組み込み,ディスプレイクローニング法によりジベレリン結合タンパク(gibberellin binding Protein : GBP)の決定を試みた。
ビオチン化GA4)をリガンドとし,イネ(Oryza sativa)の培養細胞及び出芽組織(発芽3日目,7日目)から作成したT7ファージライブラリをファージディスプレイ法でスクリーニングした。ファージとビオチン化GA4の結合を,再度ファージディスプレイ法を用いて確かめると,培養細胞ライブラリ由来の1種,出芽組織ライブラリ由来の2種のファージで結合が見られた。これら3種のファージが提示しているアミノ酸配列をDNA配列から推測すると,それぞれ,培養細胞ライブラリ由来ファージは17残基,出芽組織ライブラリ由来ファージは5残基及び10残基のペプチドを提示していた。ファージディスプレイ法で結合の最も強く見られた,培養細胞ライブラリ由来の17残基のペプチドを表面プラズモン共鳴によって結合解析をした結果,センサーチップ上に固定したペプチドとジベレリンA4の結合が確認された。この3種のペプチドのアミノ酸配列を,遺伝子データベースで検索すると,5残基で構成されるペプチド1種について,alpha-expansin2及び,nodulinと相同性が見られた。alpha-expansin2及び,nodulinをクローニングしてタンパク質を得,表面プラズモン共鳴センサーにより生体分子相互作用解析を試みようとした。しかしながら,当該遺伝子は大腸菌では全く発現しなかった。そこで,無細胞タンパク質発現系なども試みたが,事態は全く改善されていない。今後ファージディスプレイを改変した結合解析法を開発しているので,その方法を適用する予定である。

Research Products

• [Journal Article] Mammalian mitotic centromere-associated kinesin (MCAK), a new molecular target of sulfoquinovosylacylglycerols (SQAGs), novel anti-tumor and immunosuppressive agents (2005)
  • Author(s): Aoki S, Kamisuki S, Ohta S, Sugawara F, Sakaguchi K
  • Journal Title: FEBS J. (In press)
• [Journal Article] Monolayer membranes and bilayer vesicles characterized by α- and β-anomer of sulfoquinovosyldiacyglycerol (SQDG). (2005)
  • Author(s): Matsumoto.K, Sakai H, Ohta K, Kameda H, Sugawara F, Abe M, Sakaguchi K.
  • Journal Title: Chemistry and Physics of Lipids. (In press)
• [Journal Article] Affinity capture of a mammalian DNA polymerase β by inhibitors immobilized to resins used in solid-phase organic synthesis. (2005)
  • Author(s): Kuramochi K, Haruyama Y, Takeuchi R, Sunoki T, Watanabe M, Oshige M, Kobayashi S, Sakaguchi K, Sugawara F
  • Journal Title: Bioconjugate Chemistry. 16:97 Pages: 97-104
• [Journal Article] Identification of a drug target motif : an anti-tumor drug NK109 interacts with a PNxxxxP motif (2005)
  • Author(s): Kengo Morohashi, Ayako Yoshino, Atsushi Yoshimori, Seiichi Saito, Seiichi Tanuma, Kengo Sakaguchi, Fumio Sugawara
  • Journal Title: Biochem.Pharmacol. (In press)
• [Journal Article] hEP1 Receptor Third Cytoplasmic Loop : A New Target for Camptothecin Identified by Phage Display Technology (2005)
  • Author(s): Yoichi Takakusagi, Keisuke Ohta, Kouji Kuramochi, Kengo Morohashi, Susumu Kobayashi, Kengo Sakaguchi, Fumio Sugawara
  • Journal Title: Biochemistry (In press)
• [Journal Article] Design of vesicles of 1,2-di-O-acyl-3-O-(beta-D-sulfoquinovosyl)-glyceride bearing two stearic acids (beta SQDG-C(18)), a novel immunosuppressive drug. (2004)
  • Author(s): Matsumoto K, Takenouchi M, Ohta K, Ohta Y, Imura T, Oshige M, Yamamoto Y, Sahara H, Sakai H, Abe M.
  • Journal Title: Biochem Pharmacol. 68 Pages: 2379-2386
• [Journal Article] Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display. (2004)
  • Author(s): Yamazaki T, Aoki S, Ohta K, Hyuma S, Sakaguchi K, Sugawara F.
  • Journal Title: Bioorg.Med.Chem.Lett. 14 Pages: 4343-4346
• [Journal Article] Structural relationship of lithocholic acid derivatives binding to the N-terminal 8-kDa domain of DNA polymerase beta. (2004)
  • Author(s): Miura K, Hanashima S, Takemura M, Yoshida H, Sugawara F, Sakaguchi K.
  • Journal Title: Biochemistry 43 Pages: 10669-10677
• [Journal Article] Precise structural elucidation of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha (2004)
  • Author(s): Kamisuki S, Takahashi S, Mizushina Y, Sakaguchi K, Nakata T, Sugawara F
  • Journal Title: Bloorg.Med.Chem 12 Pages: 5355-5359
• [Journal Article] Molecular design of cholesterols as inhibitors of DNA polymerase alpha. (2004)
  • Author(s): Oshige M, Kuramochi K, Ohta K, Ogawa A, Kuriyama H, Sugawara F, Kobayashi S, Sakaeuchi K
  • Journal Title: J.Med.Chem. 47 Pages: 4971-4974
• [Journal Article] Structure-activity relationships of epolactaene analogs as DNA polymerases inhibitors. (2004)
  • Author(s): Kuramochi K, Mizushina Y, Nagata S, Sugawara F, Sakaguchi K, Kobayashi S.
  • Journal Title: Bloorg.Med.Chem. 12 Pages: 1983-1989
• [Journal Article] Structure-activity relationships of untenone A and its derivatives for inhibition of DNA polymerases (2004)
  • Author(s): Saito F, Takeuchi R, Kamino T, Kuramochi K, Sugawara F, Sakaguchi K, Kobayashi S.
  • Journal Title: Bloorg.Med.Chem.Lett. 14 Pages: 1975-1977
• [Journal Article] Protective properties of neoechinulin A against SIN-1-induced neuronal cell death. (2004)
  • Author(s): Maruyama K, Ohuchi T, Yoshida K, Shibata Y, Sugawara F, Arai T.
  • Journal Title: J.Biochem.(Tokyo) 36 Pages: 81-87
  • NAID: 10016200897
• [Journal Article] Synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin reduces hepatic ischemia-reperfusion injury in rats. (2004)
  • Author(s): Tsuruma T, Sahara H, Takenouchi M, Yagihashi A, Iwayama Y, Shima H, Furuhata T, Torigoe T, Hanashima S, Yamazaki T, Sugawara F, Mizushina Y, Ohta K, Sakaguchi K, Sato N, Hirata K.
  • Journal Title: Transplant Proc 36 Pages: 1965-1969
• [Journal Article] The effects of dehydroaltenusin, a novel mammalian DNA polymerase alpha inhibitor, on cell proliferation and cell cycle progression. (2004)
  • Author(s): Murakami-Nakai C, Maeda N, Yonezawa Y, Kuriyama I, Kamisuki S, Takahashi S, Sugawara F, Yoshida H, Sakaguchi K, Mizushina Y.
  • Journal Title: Biochim.Biophys.Acta. 1674 Pages: 193-199
• [Journal Article] Pseudodeflectusin, a novel isochroman derivative from Aspergillus pseudodeflectus a parasite of the sea weed, Sargassum fusiform, as a selective human cancer cytotoxin (2004)
  • Author(s): Ogawa A, Murakami C, Kamisuki S, Kuriyama I, Yoshida H, Sugawara F, Mizushina Y.
  • Journal Title: Bioorg.Med.Chem.Lett. 14 Pages: 3539-3543
• [Journal Article] Taxol derivatives are selective inhibitors of DNA polymerase alpha. (2004)
  • Author(s): Oshige M, Takenouchi M, Kato Y, Kamisuki S, Takeuchi T, Kuramochi K, Shiina I, Suenaga Y, Kawakita Y, Kuroda K, Sato N, Kobayashi S, Sugawara F, Sakaguchi K.
  • Journal Title: Bioorg.Med.Chem. 12 Pages: 2597-2601
• [Patent(Industrial Property Rights)] 新規イソクロマノン化合物とその行為丸剤への利用 (2004)
  • Inventor(s): 菅原 二三男, 水品 善之 他
  • Industrial Property Rights Holder: 東京理科大学
  • Filing Date: 2004-04-19
• [Patent(Industrial Property Rights)] 新規な放射線治療増感剤 (2004)
  • Inventor(s): 菅原 二三男, 山崎 隆之 他
  • Industrial Property Rights Holder: 東京理科大学
  • Filing Date: 2004-06-24
• [Patent(Industrial Property Rights)] Administration of a sulfopyranosylacylglycerol to treat certain cancer (2004)
  • Inventor(s): 菅原 二三男, 山崎 隆之 他
  • Industrial Property Rights Holder: 東洋水産(株)
  • Acquisition Date: 2004-08-03
• [Publications] Mizushina Y, Maeda N, Kawasaki M, Ichikawa H, Murakami C, Takemura M, Xu X, Sugawara F, Fukumori Y, Yoshida H, Sakaguchi K.: "Inhibitory action of emulsified sulfoquinovosyl acylglycerol on mammalian DNA polymerases."Lipids. 33. 1065-1074 (2003)
• [Publications] Mizushina Y, Hirota M, Murakami C, Ishidoh T, Kamisuki S, Shimazaki N, Takemura M, Perpelescu M, Suzuki M, Yoshida H, Sugawara F, Koiwai O, Sakaguchi K.: "Some anti-chronic inflammatory compounds are DNA polymerase lambda-specific inhibitors."Biochem.Pharmacol.. 66. 1935-1944 (2003)
• [Publications] Kimura S, Furukawa T, Kasai N, Mori Y, Kitamoto HK, Sugawara F, Hashimoto J, Sakaguchi K.: "Functional characterization of two flap endonuclease-1 homologues in rice."Gene. 314. 63-71 (2003)
• [Publications] Murakami C, Takemura M, Yoshida H, Sugawara F, Sakaguchi K, Mizushina Y.: "Analysis of cell cycle regulation by 1-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride (SQMG), an inhibitor of eukaryotic DNA polymerases."Biochem.Pharmacol.. 66. 541-550 (2003)
• [Publications] Namekawa S, Ichijima Y, Hamada F, Kasai N, Iwabata K, Nara T, Teraoka H, Sugawara F, Sakaguchi K.: "DNA ligase IV from a basidiomycete, Coprinus cinereus, and its expression during meiosis."Microbiology. 149. 2119-2128 (2003)
• [Publications] Mizushina Y, Murakami C, Takikawa H, Kasai N, Xu X, Mori K, Oshige M, Yamaguchi T, Saneyoshi M, Shimazaki N, Koiwai O, Yoshida H, Sugawara F, Sakaguchi K.: "Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos."Biochem.Biophys.Res.Commun.. 305. 365-373 (2003)