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Development of the system for a large-scale expression of membrane proteins towards their crystallization.

Research Project

Project/Area Number 10680632
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionNagoya University

Principal Investigator

SUGIYAMA Yasuo  Nagoya University, Institute for Gene Research, Associate Professor, 遺伝子実験施設, 助教授 (70154507)

Co-Investigator(Kenkyū-buntansha) IHARA Kunio  Nagoya University, Institute for Gene Research, Assistant Professor, 遺伝子実験施設, 助手 (90223297)
KUSUMI Akihiro  Nagoya University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (50169992)
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsRetinal proteins / light-driven ion pump / heterologous expression ysytem / fission yeast / GFP-fusion protiens / two-dimensional crystal / tubular structure / 光センサー / (His)6-Tagタンパク質
Research Abstract

We have examined the methods to isolate large amounts of intact retinal proteins expressed in Schizoseaccharomyces pombe. Retinal proteins with (His)6-Tag at a C-terminal were expressed in S.pombe, however, they could not be purified by Ni-NTA agarose column chromatography. Therefore, it was concluded that (His) 6-Tag method does not replace the conventional one.
Retinal proteins with GFP at a C-terminal were expressed and distributed in the membranes of ER and vacuole in S.pombe at a log phase. At a stationary phase, they were digested and released GFP were distributed in cytoplasm and nuclear membrane. Therefore, it is necessary to disrupt the cells immediately after expression the proteins.
We found that archaerhodopsin (aR) forms two-dimensional crystal in the membrane as bacteriorhodopsin does and that aR transforms from membrane sheet to tubular structure when stored in a refrigerator. We are going to find out the condition to crystallize aR.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Kunio Ihara: "Evolution of the archaeal rhodopsins : Evolution rate changes by gene duplication andfunctional differentiation."J.Mol.Biol.. 285. 163-174 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yasuo Mukohata: "Halobacterial Rhodopsins."J.Biochem.. 125. 649-657 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yasuo Sugiyama: "The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin."J.Biochem.. 125. 1144-1150 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kunio Ihara: "Evolution of the archaeal rhodopsins : Evolution rate changes by gene duplication and functional differentiation."J.Mol.Biol.. 285. 163-174 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yasuo Mukohata: "Halobacterial Rhodopsins"J.Biochem.. 125. 649-657 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yasuo Sugiyama: "The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin."J.Biochem.. 125. 1144-1150 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kunio Ihara: "Evolution of the archaeal rhodopsins : Evolution rate changes by gene duplication andfunctional differentiation."J.Mol.Biol.. 285. 163-174 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yasuo Mukohata: "Halobacterial Rhodopsins."J.Biochem.. 125. 649-657 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yasuo Sugiyama: "The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin."J.Biochem.. 125. 1144-1150 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kunio Ihara他: "Evoloution of the archaeal rhodopsins : Evolution rate changes by gene duplication and functional differentiatiou." J.Mol.Biol.285. 163-174 (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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