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Struchural biology of membrane protein trabsporters

Research Project

Project/Area Number 17380066
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied biochemistry
Research InstitutionKyoto University

Principal Investigator

KATO Hiroaki  Kyoto University, Graduate School of Pharmaceutical Sciences, Professor (90204487)

Co-Investigator(Kenkyū-buntansha) NAKATSU Toru  Kyoto University, Graduate School of Pharmaceutical Sciences, Associate Professor (50293949)
NAKANO Hiroaki  Kyoto Universtiy, Graduate School of Pharmaceutical Sciences, assistant professor (10378789)
KODAN Atsushi  Kyoto Universty, Graduate School of Medicine, Researcher (80360543)
MITSUOKA Kaoru  National Instifute of Advanced Industrial Science and Technology, Chief Researcher (60301230)
Project Period (FY) 2005 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥15,480,000 (Direct Cost: ¥14,700,000、Indirect Cost: ¥780,000)
Fiscal Year 2007: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
Fiscal Year 2006: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥8,500,000 (Direct Cost: ¥8,500,000)
KeywordsABC transporter / P-glycoprotein / X-ray crystalloqraphy / Single Particle Analysis / membrane proteins / トランスポーター / 構造生物学 / 電子顕微鏡 / 多剤耐性 / 胆汁排出 / 糖尿病
Research Abstract

We gained the following research results :
1. Multi-drug resistance transporter MDR1 from human was overexpressed and purified using Sf^+ insect cell-baculovirus expression system. The purified MDR1 showed a single band on SDS-PAGE. An E. coli homologue of MDR1, MsbA was also expressed and purified. MsbA was crystallized and diffracted by X-ray with 7 A resolution.
2. Bile salt export pump (BSEP) from rat was overexpressed and purified in the Sf^+ cells. Some homologues of BSEP were cloned from thermophilic microorganisms and purified for crystallization experiments. A clone suitable for the crystallization work.
3. A putative very long chain fatty acid transporter, PMP70 was expressed in Pichia pastoris. However, the expression level was not enough to do crystallization experiments. Thus, we have done characterization of Pex19p that participates PMP70 translocation into peroxisomal membrane, and its receptor Pex3p.
4. KATP channel, SUR1-Kir6.2 complex was co-expressed in HEK293 cells. The obtained cells showed KATP channel activily regulated by its blocker regents. The SUR1-Kir6.2 complex was purified as a single band on SDS-PAGE. The structure detemination of the purified preparation started by single particle analysis using cryo-electron microscopy.
5. ATP is the important energy source of ABC transporters but its role in the transporter mechanism is still unclear because ATP complex structure of ABC transporter has not solved yet. Thus, we solved the firefly luciferase structure complexed with its reaction intermediate analogue. Firefly luciferase needs ATP to activate the reactivity of the substrate, luciferin. The structure showed us structural-basis of the reaction, expecialy how ATP assists the reaction.

Report

(4 results)
  • 2007 Annual Research Report   Final Research Report Summary
  • 2006 Annual Research Report
  • 2005 Annual Research Report
  • Research Products

    (39 results)

All 2008 2007 2006 2005

All Journal Article (20 results) (of which Peer Reviewed: 8 results) Presentation (18 results) Book (1 results)

  • [Journal Article] Characterization of the interaction between Recombinant Human Peroxin Pex3p and Pex19p2008

    • Author(s)
      Yasuhiko Sato
    • Journal Title

      Journal of Biological Chemistry 283

      Pages: 6136-6144

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Characterization of the Interaction between Recombinant Human Peroxin Pex3_p and. Pex 19_p : IDENTIFICATION OF TRP-104 IN Pex3_p AS A CRITICAL RESIDUE FOR THE INTERACTION2008

    • Author(s)
      Sato, Y
    • Journal Title

      J. Biol. Chem 283

      Pages: 6136-6144

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Crystallization and preliminary Y-ray crystallographic studies of Pz peptidase A from Geobacillus collagenovorans MO-1.2007

    • Author(s)
      Nakano Hitoaki
    • Journal Title

      Acta Crystallographica F63

      Pages: 142-144

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol2007

    • Author(s)
      Yasuhisa Kimura
    • Journal Title

      Biochemical Journal 401

      Pages: 597-605

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Crystallization and preliminary X-ray crystallographic studies of P_z peptidase A from Geobacillus collagenovorans MO-12007

    • Author(s)
      Kawasaki, A
    • Journal Title

      Acta Crystallogr 63

      Pages: 142-144

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol2007

    • Author(s)
      Kimura, Y
    • Journal Title

      Biochem. J 401

      Pages: 597-605

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] 1. Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol2007

    • Author(s)
      木村泰久
    • Journal Title

      Biochemical Journal 401・2

      Pages: 597-605

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Overexpression, purification, crystallization and preliminary X-raycrystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1.2006

    • Author(s)
      Makoto Akioka
    • Journal Title

      Acta Crystallographica F62

      Pages: 1266-1268

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Structural basis for the spectral difference in luciferase bioluminescence.2006

    • Author(s)
      Toru Nakatsu
    • Journal Title

      Nature 440

      Pages: 372-376

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Overexpression, purification, crystallization and preliminary X-ray crystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-12006

    • Author(s)
      Akioka, M.
    • Journal Title

      Acta Crystallogr 62

      Pages: 1266-11268

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Structural basis for the spectral difference in luciferase bioluminescence2006

    • Author(s)
      Nakatsu, T
    • Journal Title

      Nature 440

      Pages: 372-376

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Role of Pex19p in the targeting of PMP70 to peroxisome.2005

    • Author(s)
      Yoshinori Kashiwayama
    • Journal Title

      Biochimica et Biophysica Acta 1746

      Pages: 116-128

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli.2005

    • Author(s)
      Tetsuva Shimizu
    • Journal Title

      Protein expression and purification 44(2)

      Pages: 130-135

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Crystallization and preliminary crystallographic analysis of bacterial fructosyl amino acid oxidase.2005

    • Author(s)
      Sakaue, R
    • Journal Title

      Acta Crystallographica F61

      Pages: 196-198

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Role of Pex 19_p in the targeting of PMP70 to peroxisome2005

    • Author(s)
      Kashiwayama, Y
    • Journal Title

      Biochim. Biophys. Acta 1746

      Pages: 116-128

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli2005

    • Author(s)
      Shimizu, T
    • Journal Title

      Protein Expr. Purif 44(2)

      Pages: 130-135

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Crystallization and preliminary crystallographic analysis of bacterial fructosyl amino acid oxidase2005

    • Author(s)
      Sakaue, R
    • Journal Title

      Acta Crystallogr 61

      Pages: 196-198

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Role of Pex19p in the targeting of PMP70 to peroxisome2005

    • Author(s)
      Kashiwayama, Y.
    • Journal Title

      Biochim.Biophys.Acta 1746

      Pages: 116-128

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli2005

    • Author(s)
      Shimizu, T.
    • Journal Title

      Protein Expression and Purification 44・2

      Pages: 130-135

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Crystallization and preliminary crystallographic analysis of bacterial fructosyl amino acid oxidase.2005

    • Author(s)
      Sakaue, R.
    • Journal Title

      Acta Crystallogr. F61

      Pages: 196-198

    • Related Report
      2005 Annual Research Report
  • [Presentation] Screening of suitable thermophiles' ABC transporter for crystallization2008

    • Author(s)
      Tomomi, Sato
    • Organizer
      The 128th annual meeting of Pharmaceutical Society of Japan
    • Place of Presentation
      Pacifico Yokohama (Yokohama)
    • Year and Date
      2008-05-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Screening of eukaryotic ABC transporter for structural analysis2008

    • Author(s)
      Keita, Sakiyama
    • Organizer
      The 128th annual meeting of Pharmaceutical Society of Japan
    • Place of Presentation
      Pacifico Yokohama (Yokohama)
    • Year and Date
      2008-05-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 構造解析に適した真核生物由来のABCトランスポーターのスクリーニング2008

    • Author(s)
      崎山慶太
    • Organizer
      日本薬学会第128年会
    • Place of Presentation
      パシフィコ横浜(横浜市)
    • Year and Date
      2008-03-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
  • [Presentation] 結晶化に適した好熱菌由来ABCトランスポーターのスクリーニング2008

    • Author(s)
      佐藤友美
    • Organizer
      日本薬学会第128年会
    • Place of Presentation
      パシフィコ横浜(横浜市)
    • Year and Date
      2008-03-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 結晶化に適した好熱菌由来ABCトランスポーターのスクリーニング2008

    • Author(s)
      佐藤友美
    • Organizer
      日本薬学会第128年会
    • Place of Presentation
      パシフィコ横浜(横浜布)
    • Year and Date
      2008-03-28
    • Related Report
      2007 Annual Research Report
  • [Presentation] 結晶化を目指したペルオキシソーム膜タンパク質22のメタノール資化性酵母Pichia pastorisにおける大量生産2007

    • Author(s)
      江川響子
    • Organizer
      第7回日本蛋白質科学会年会
    • Place of Presentation
      仙台国際センター(仙台)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 多剤排出トランスポーターP糖タンパク質におけるリンカー領域の役割2007

    • Author(s)
      佐藤友美
    • Organizer
      第7回日本蛋白質科学会年会
    • Place of Presentation
      仙台国際センター(仙台)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] メタノール資化性酵母Pichia pastorisを用いたラットペルオキシソーム膜タンパク質22の発現系の構築2007

    • Author(s)
      江川響子
    • Organizer
      日本薬学会第127年会
    • Place of Presentation
      富山大学(富山)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Overproduction of peroxisomal membrane protein 22 in methylotrophic yeast Pichia pastoris for crystallization2007

    • Author(s)
      Kyoko, Egawa
    • Organizer
      The 7th annual meeting of the protien science society of Japan
    • Place of Presentation
      Sendai international center (Sendai)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Role of the linker region of multidrug efflux transporter P-glycoprotein2007

    • Author(s)
      Tomomi, Sato
    • Organizer
      The 7th annual meeting of the protein science society of Japan
    • Place of Presentation
      Sendai international center (Sendai)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Expression of rat Peroxisomal Membrane Protein Methylotrophic Yeast by Pichia pastoris2007

    • Author(s)
      Kyoko, Egawa
    • Organizer
      The 127th annual meeting of the Pharmaceutical Society of Japan
    • Place of Presentation
      Toyama University (Toyama)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Overproduction of rattus peroxisomal membrane protein 22 in Pichia Pastoris2006

    • Author(s)
      Kyoko Egawa
    • Organizer
      Pichia protein expression conference
    • Place of Presentation
      San Diego, USA
    • Year and Date
      2006-10-11
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 真菌Stereum purpureum由来エンドポリガラクツロナーゼの超高分解能(0.62Å)X線結晶構造解析2006

    • Author(s)
      清水哲哉
    • Organizer
      第6回日本蛋白質科学会年会
    • Place of Presentation
      京都国際会館(京都市)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Ultra-high resolution (0.62 A) X-ray crystallography of endopolygalact uronase from Stereum purpureum2006

    • Author(s)
      Tetsuya, Shimizu
    • Organizer
      The 6th Annual Meeting of the Protein Science Society of Japan
    • Place of Presentation
      Kyoto Internationnal Conference Hall (Kyoto)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Large scale purification of P-glycoprotein from Sf+ insect cells2005

    • Author(s)
      Tomomi, Sato
    • Organizer
      27th Symposium on Biomembrane-Drug Interaction
    • Place of Presentation
      Kyoto, Japan
    • Year and Date
      2005-11-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] リンゴ銀葉病菌由来エンドポリガラクツロナーゼの超高分解能X線結晶構造解析と高分解能中性子結晶構造解析2005

    • Author(s)
      清水哲哉
    • Organizer
      日本結晶学会2005年年会
    • Place of Presentation
      イーグレひめじ(姫路市)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 昆虫細胞Sf+を用いたヒトP糖タンパク質の大量調製2005

    • Author(s)
      佐藤友美
    • Organizer
      第27回生体膜と薬物の相互作用シンポジウム
    • Place of Presentation
      京都大学医学部創立百周年記念施設「芝蘭会館」(京都市)
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Ultra-high resolution X-ray and neutron crystallography of Stereum purpureum endopolygalacturonase2005

    • Author(s)
      Tetsuya, Shimizu
    • Organizer
      Annual Meeting 2005
    • Place of Presentation
      Egret Himeji (Himeji)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Book] ABC蛋白質の立体構造研究-現状と課題-ABC蛋白質(植田和光編)2005

    • Author(s)
      加藤 博章
    • Total Pages
      282
    • Publisher
      学会出版センター
    • Related Report
      2005 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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