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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

Research Project

Project/Area Number 15651101
Research Category

Grant-in-Aid for Exploratory Research

Allocation TypeSingle-year Grants
Research Field Living organism molecular science
Research InstitutionTokyo 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 *help
¥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。

Report

(2 results)
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (26 results)

All 2005 2004 Other

All Journal Article (17 results) Patent(Industrial Property Rights) (3 results) Publications (6 results)

  • [Journal Article] Mammalian mitotic centromere-associated kinesin (MCAK), a new molecular target of sulfoquinovosylacylglycerols (SQAGs), novel anti-tumor and immunosuppressive agents2005

    • Author(s)
      Aoki S, Kamisuki S, Ohta S, Sugawara F, Sakaguchi K
    • Journal Title

      FEBS J. (In press)

    • Related Report
      2004 Annual Research Report
  • [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)

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Identification of a drug target motif : an anti-tumor drug NK109 interacts with a PNxxxxP motif2005

    • Author(s)
      Kengo Morohashi, Ayako Yoshino, Atsushi Yoshimori, Seiichi Saito, Seiichi Tanuma, Kengo Sakaguchi, Fumio Sugawara
    • Journal Title

      Biochem.Pharmacol. (In press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] hEP1 Receptor Third Cytoplasmic Loop : A New Target for Camptothecin Identified by Phage Display Technology2005

    • Author(s)
      Yoichi Takakusagi, Keisuke Ohta, Kouji Kuramochi, Kengo Morohashi, Susumu Kobayashi, Kengo Sakaguchi, Fumio Sugawara
    • Journal Title

      Biochemistry (In press)

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Precise structural elucidation of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha2004

    • Author(s)
      Kamisuki S, Takahashi S, Mizushina Y, Sakaguchi K, Nakata T, Sugawara F
    • Journal Title

      Bloorg.Med.Chem 12

      Pages: 5355-5359

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Structure-activity relationships of untenone A and its derivatives for inhibition of DNA polymerases2004

    • 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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Pseudodeflectusin, a novel isochroman derivative from Aspergillus pseudodeflectus a parasite of the sea weed, Sargassum fusiform, as a selective human cancer cytotoxin2004

    • 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

    • Related Report
      2004 Annual Research Report
  • [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

    • Related Report
      2004 Annual Research Report
  • [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
    • Related Report
      2004 Annual Research Report
  • [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
    • Related Report
      2004 Annual Research Report
  • [Patent(Industrial Property Rights)] Administration of a sulfopyranosylacylglycerol to treat certain cancer2004

    • 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
    • Related Report
      2004 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report
  • [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)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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