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Creation of artificial peptidase based on zinc finger framework

Research Project

Project/Area Number 20510206
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Living organism molecular science
Research InstitutionDoshisha Women's College of Liberal Arts

Principal Investigator

NEGI Shigeru  Doshisha Women's College of Liberal Arts, 薬学部, 助教 (50378866)

Project Period (FY) 2008 – 2010
Project Status Completed (Fiscal Year 2010)
Budget Amount *help
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2009: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2008: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
Keywords蛋白質 / 生体機能利用 / 生体分子 / 核酸・薬学 / 亜鉛フィンガー / 金属フィンガー / ペプチドデザイン / 人工制限酵素 / 人工ペプチダーゼ / Sp1亜鉛フィンガー / GAGAファクター / 転写因子 / Spl亜鉛フィンガー
Research Abstract

Zinc finger protein is naturally occurring DNA-binding protein found in many transcription factors. Zinc finger protein has two precious molecular recognition abilities ; (1) metal ion binding and (2) DNA-binding abilities with high affinity and selectivity. Because of these characteristic, zinc finger protein is promising framework for designing artificial zinc finger proteins. In this project, I tried to introduce the catalytic activities, hydrolytic, nuclease and peptidase activities, to Sp1 zinc finger protein itself by redesign of metal binding site. At first, three His4-type zinc finger domains (FxH4, x=1,2 and 3) were created by mutation of two Cys residues to His residues for each three zinc finger domain of Sp1. All His4-type zinc finger peptides possess the high hydrolytic activities for BNPP having phosphate ester bond. Especially, F1H4 has much higher hydrolytic activity for phosphate ester compare to that of F2H4 and F3H4. Next, new types of fusionl zinc finger nuclease, ZWH4 and Sp1fxH4Sp1, were prepared. ZWH4 was created by connecting F2H4 domain to c-terminus of Sp1 zinc finger protein. ZWH4 could efficiently cleavage plasmid pUC19 DNA containing target sequence. In addition, ZWH4 showed also peptidase activity. In order to increase the binding affinity for target DNA sequence and the catalytic activity of Hie4-type zinc finger protein, Sp1FxH4Sp1 (x=1 and 2), 7-finger proteins, were created by combine wild-type Sp1 to both N- and C-terminus of FxH4 zinc finger domain. Sp1FxH4Sp1 can produce as His-tag fusion protein using E coli protein expression systems. In gel mobility shift assay, Sp1FxH4Sp1 could bind to target DNA sequence. Moreover, in preliminary DNA cutting experiment, Sp1F2H4Sp1 showed week hydrolytic activity for plasmid DNA.

Report

(4 results)
  • 2010 Annual Research Report   Final Research Report ( PDF )
  • 2009 Annual Research Report
  • 2008 Annual Research Report
  • Research Products

    (16 results)

All 2011 2010 2009 2008 Other

All Journal Article (8 results) (of which Peer Reviewed: 8 results) Presentation (6 results) Book (2 results)

  • [Journal Article] Zinc finger-zinc finger interaction between the transcription factors, GATA-land Sp12010

    • Author(s)
      S.Negi
    • Journal Title

      Biochem.Biophys.Res.Commun

      Volume: 400 Pages: 625-630

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cytochrome C polymerization by successive domain swapping at the C-termina helix625-6302010

    • Author(s)
      S.Negi
    • Journal Title

      Proc.Natl.Acad.Sci.USA.

      Volume: 107 Pages: 12854-12859

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A diatomic molecule receptor that removes CO in a living organism2010

    • Author(s)
      S.Negi
    • Journal Title

      Angew.Chem.Int.Ed.

      Volume: 49 Pages: 1312-1315

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Novel zinc finger nuclease created by combining the Cys_2His_2- and His_4-type zinc finger domains2009

    • Author(s)
      Shigeru Negi, Yoshiyuki Umeda, Saeko Masuyama Saeko, Koji Kano, Yukio Sugiura
    • Journal Title

      Bioorganic & Medicinal Chemistry Letter Vol.19

      Pages: 2789-2791

    • Related Report
      2010 Final Research Report
    • Peer Reviewed
  • [Journal Article] Novel zinc finger nuclease Created by combining the Cys_2His_2 and His_4 type zinc finger domain2009

    • Author(s)
      S.Negi
    • Journal Title

      Bioorg.Med.Chem.Lett. 19

      Pages: 2789-2791

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Effects of Bulkiness and Hydrophobicity of an Aliphatic Amino Acid in the Recognition2008

    • Author(s)
      Muthu Dhanasekaran, Shigeru Negi, Miki Imanishi, Michiko Suzuki, Yukio Sugiura
    • Journal Title

      Helix of the GAGA Zinc Finger on the Stability of the Hydrophobic Core and DNA Binding Affinity, Biochemistry Vol.47

      Pages: 11717-11724

    • Related Report
      2010 Final Research Report
    • Peer Reviewed
  • [Journal Article] Effects of bulkiness and hydrophobicity of an aliphatic amimo acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding atrinily2008

    • Author(s)
      M. Dhanasekaran
    • Journal Title

      Biochemistry 47

      Pages: 11717-11724

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] New redesigned zinc-finger proteins : design strategy and its application2008

    • Author(s)
      S. Negi
    • Journal Title

      Chemistry 14

      Pages: 3236-3246

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Presentation] Redesigned Zinc Finger Proteins : Design Strategy and Its Application2011

    • Author(s)
      S.Negi
    • Organizer
      Pepcom2011
    • Place of Presentation
      北京
    • Year and Date
      2011-03-23
    • Related Report
      2010 Annual Research Report
  • [Presentation] DNA結合におけるGAGA亜鉛フィンガードメイン疎水性コアの重要性2009

    • Author(s)
      根木滋
    • Organizer
      日本化学会第89回春季年会(2009)
    • Place of Presentation
      日本大学
    • Year and Date
      2009-03-29
    • Related Report
      2008 Annual Research Report
  • [Presentation] Non FokI-type zinc finger nuclease2009

    • Author(s)
      根木滋
    • Organizer
      14^<th> International Conference on Biological Inorganic Chemistry
    • Place of Presentation
      名古屋
    • Related Report
      2009 Annual Research Report
  • [Presentation] Non FokI-type zinc finger nuclease2008

    • Author(s)
      Shigeru Negi, Yukio Sugiura
    • Organizer
      14th International Conference on Biological Inorganic Chemistry (ICBIC14)
    • Place of Presentation
      Nagoya
    • Related Report
      2010 Final Research Report
  • [Presentation] Redesigned Zinc Finger Proteins : Design Strategy and Its Application

    • Author(s)
      Shigeru Negi, Yukio Sugiura
    • Organizer
      Pepcom2011 (Beijing)
    • Place of Presentation
      Invited Lecture
    • Related Report
      2010 Final Research Report
  • [Presentation] Design of artificial zinc finger nuclease based on unnatural His4-type zinc finger catalytic domain

    • Author(s)
      Shigeru Negi, Yukio Sugiura
    • Organizer
      Pacifichem 2010
    • Place of Presentation
      Hawaii
    • Related Report
      2010 Final Research Report
  • [Book] "Non-FokI-based zinc finger nuclease" in Engineered Zinc Finger proteins2010

    • Author(s)
      S.Negi
    • Total Pages
      12
    • Publisher
      Methods in Molecular Biology, Vol.649
    • Related Report
      2010 Annual Research Report
  • [Book] Non-FokI-based zinc finger nuclease(Engineered Zinc Finger proteins, <Eds. by J. Mackay and D. Segal>, Methods in Molecular Biology, Vol.649)

    • Author(s)
      Shigeru Negi, et. al.
    • Related Report
      2010 Final Research Report

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Published: 2008-04-01   Modified: 2016-04-21  

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