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Screening of a minimal reporter protein with correct SS-bonds using a novel VanX mediated E-coli autolysis protocol

Research Project

Project/Area Number 26560432
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Biomolecular chemistry
Research InstitutionTokyo University of Agriculture and Technology

Principal Investigator

Kuroda Yutaka  東京農工大学, 工学(系)研究科(研究院), 准教授 (10312240)

Project Period (FY) 2014-04-01 – 2016-03-31
Project Status Completed (Fiscal Year 2015)
Budget Amount *help
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2014: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Keywords蛋白質工学 / 生物活性物質の探索 / SS結合 / 探索法 / ランダム変異 / VanX / 溶菌 / 生物発光
Outline of Final Research Achievements

Gaussia Luciferase (GLuc; 168 aa) is among the smallest luciferase and could serve as a reported protein in biochemical research. Its identification is relatively recent, and its structure and biophysical properties remain to be fully characterized. Here, we developed a novel method for screening GLuc, where VanX is coexpressed with Gluc in order to autolyse E-coli and release GLuc into the culture media. This, in turn, enables GLuc’s bioluminescence measurement directly in the crude media without purification greatly increasing the screening efficiency. Using this protocol we identified several single mutation GLuc variants with light emission red shift by up to 5 nm, and a double mutation variant with red shift of 15 nm (but with weaker light emission intensity). In addition to this, we started a heteronuclear NMR analysis of recombinant GLuc, and we assigned about 70% of its main chain peak and their secondary structures.

Report

(3 results)
  • 2015 Annual Research Report   Final Research Report ( PDF )
  • 2014 Research-status Report
  • Research Products

    (31 results)

All 2016 2015 2014

All Journal Article (9 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 8 results,  Acknowledgement Compliant: 4 results,  Open Access: 2 results) Presentation (22 results) (of which Int'l Joint Research: 1 results,  Invited: 6 results)

  • [Journal Article] A novel screening system based on VanX mediated autolysis - application to Gaussia luciferase2016

    • Author(s)
      Wu Nan, Kamioka Tetsuya, Kuroda Yutaka
    • Journal Title

      Biotechnol Bioeng

      Volume: 印刷中 Issue: 7 Pages: 1413-1420

    • DOI

      10.1002/bit.25910

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants2015

    • Author(s)
      Kulkarni R Manjiri, Islam M Mohammad, Numoto Nobutaka, Elahi Montasir, Mahib R Mamunur, Ito Nobutoshi, Kuroda Yutaka
    • Journal Title

      Biochim Biophys Acta

      Volume: 1854 Issue: 10 Pages: 1438-43

    • DOI

      10.1016/j.bbapap.2015.07.004

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Analysis and control of protein crystallization using short peptide tags that change solubility without structure, thermal stability and function2015

    • Author(s)
      Islam M, Nakamura S, Noguchi K, Yohda M, Kidokoro S, Kuroda Y
    • Journal Title

      Crystal Growth Design

      Volume: 15 Issue: 6 Pages: 2703-2711

    • DOI

      10.1021/acs.cgd.5b00010

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Bacterial expression and re-engineering of Gaussia princeps luciferase and its use as a reporter protein2015

    • Author(s)
      Wu Nan, Rathnayaka Tharangani, Kuroda Yutaka
    • Journal Title

      Biochim Biophys Acta

      Volume: 1854 Issue: 10 Pages: 1392-9

    • DOI

      10.1016/j.bbapap.2015.05.008

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] 蛍光セルフクエンチを用いたアモルファス凝集形成機構の解析2015

    • Author(s)
      如澤浩樹, 座古保, 前田瑞夫, 千葉一裕, 黒田裕
    • Journal Title

      月刊バイオインダストリー(シーエムシー出版)

      Volume: 9月号 Pages: 36-41

    • Related Report
      2015 Annual Research Report
  • [Journal Article] Plant-PrAS: A database of physicochemical and structural properties, and novel functional regions in plant proteomes2015

    • Author(s)
      Atsushi Kurotani, Yutaka Yamada, Kazuo Shinozaki, Yutaka Kuroda, and Tetsuya Sakurai
    • Journal Title

      Plant and Cell Physiology

      Volume: 56(1) Issue: 1 Pages: e11-e11

    • DOI

      10.1093/pcp/pcu176

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Biochemical and biophysical characterization of an unexpected bacteriolysis activity of VanX- a member of the vancomycin-resistance vanA gene cluster-2014

    • Author(s)
      Shihori Sohya, Tetsuya Kamioka, Chisako Fujita, Tei Maki, Yoshihiro Ohta, and Yutaka Kuroda
    • Journal Title

      J Biol, Chem

      Volume: 289(52) Issue: 52 Pages: 35686-35694

    • DOI

      10.1074/jbc.m114.590265

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] H-DROP: an SVM based helical domain linker predictor trained with features optimized by combining random forest and stepwise selection2014

    • Author(s)
      Teppei Ebina, Suzuki Ryosuke, Ryotaro Tsuji, Yutaka Kuroda
    • Journal Title

      J Comput Aided Mol Des

      Volume: 28(8) Issue: 8 Pages: 831-839

    • DOI

      10.1007/s10822-014-9763-x

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Correlations between protein disorder and post-translational modifications in plants2014

    • Author(s)
      3. Kurotani A, Tokmakov AA, Kuroda Y, Fukami Y, Shinozaki K, Sakurai T.
    • Journal Title

      Bioinformatics

      Volume: v. 30 Issue: 8 Pages: 1095-1103

    • DOI

      10.1093/bioinformatics/btt762

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access
  • [Presentation] Plant-PrAS: a database of physicochemical and structural properties and the comparative analysis in plant proteomes2016

    • Author(s)
      Atsushi Kurotani, Alexander Tokmakov, Yutaka Kuroda, Kazuo Shinozaki, Tetsuya Sakurai
    • Organizer
      第57回植物生理学会年会
    • Place of Presentation
      岩手大学上田キャンパス(岩手県盛岡市)
    • Year and Date
      2016-03-18
    • Related Report
      2015 Annual Research Report
  • [Presentation] ペプチド溶解性の分子動力学シミュレーション2016

    • Author(s)
      黒田 裕
    • Organizer
      第5回日本生物物理学会関東支部会
    • Place of Presentation
      (財)桐生地域地場産業振興センター(群馬県桐生市)
    • Year and Date
      2016-03-10
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] 分子動力学シミュレーションを用いたタンパク質・ペプチドの溶解性解析2016

    • Author(s)
      黒田 裕
    • Organizer
      大阪大学蛋白室研究所セミナー「構造を基盤とする蛋白質科学における未解決問題」
    • Place of Presentation
      東京大学先端科学技術研究センター(東京都目黒区)
    • Year and Date
      2016-03-01
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] Modeling and experimental assessment of buried Leu-Ile mutation in DEN4 ED32016

    • Author(s)
      Kulkarni Manjiri Ravindra, Nobutaka Numoto, Nobutoshi Ito, Yutaka Kuroda
    • Organizer
      大阪大学蛋白室研究所セミナー「構造を基盤とする蛋白質科学における未解決問題」
    • Place of Presentation
      東京大学先端科学技術研究センター(東京都目黒区)
    • Year and Date
      2016-03-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] 格子モデルを用いたタンパク質凝集のシミュレーション2016

    • Author(s)
      河村 直樹, 鈴木 涼祐, 黒田 裕
    • Organizer
      大阪大学蛋白室研究所セミナー「構造を基盤とする蛋白質科学における未解決問題」
    • Place of Presentation
      東京大学先端科学技術研究センター(東京都目黒区)
    • Year and Date
      2016-03-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] 機械学習法SVMを用いたヘリカルリンカー予測機H-DROP2016

    • Author(s)
      井出 宗一郎, Tambi Richa, 鈴木 涼祐, 蝦名 鉄平, 黒田 裕
    • Organizer
      大阪大学蛋白室研究所セミナー「構造を基盤とする蛋白質科学における未解決問題」
    • Place of Presentation
      東京大学先端科学技術研究センター(東京都目黒区)
    • Year and Date
      2016-03-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] 全原子 MD シミュレーションによるアラニンスキャニング変異を導入したアミロイド形成ペプチドの凝集解析2015

    • Author(s)
      小須田 慧司, 末永 敦, 森本 元太郎, 泰地 真弘人, 黒田 裕
    • Organizer
      第53回日本生物物理学会年会
    • Place of Presentation
      金沢大学角間キャンパス(石川県金沢市)
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
  • [Presentation] 多数ペプチドから成る系の全原子分子動力学シミュレーションによるペプチド溶解性2015

    • Author(s)
      黒田 裕, 佐藤 雄士, 末永 敦, 泰地 真弘人
    • Organizer
      第53回日本生物物理学会年会
    • Place of Presentation
      金沢大学角間キャンパス(石川県金沢市)
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
  • [Presentation] Control of protein aggregation and oligomerization using short SEP (Solubility Enhancing Peptide) tags2015

    • Author(s)
      Kabir Md. Golam, Islam Mohammmad Monirul, 黒田 裕
    • Organizer
      第53回日本生物物理学会年会
    • Place of Presentation
      金沢大学角間キャンパス(石川県金沢市)
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
  • [Presentation] Accelerated H-DROP: An SVM based Helical Domain linker pRedictor trained with OPtimized features2015

    • Author(s)
      Tambi Richa, Ide Soichiro, Suzuki Ryosuke, Ebina Teppei, Kuroda Yutaka
    • Organizer
      第53回日本生物物理学会年会
    • Place of Presentation
      金沢大学角間キャンパス(石川県金沢市)
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] Peptides solubility estimated by all-atom molecular dynamics simulation of multipeptide systems concur with experimental values2015

    • Author(s)
      Yutaka Kuroda
    • Organizer
      IUPAC 45th World Chemistry Congress
    • Place of Presentation
      釜山(韓国)
    • Year and Date
      2015-08-07
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 複数ペプチドの全原子分子動力学シミュレーションを用いたアミロイド形成機構の解析2015

    • Author(s)
      小須田慧司, 末永敦, 泰地真弘人, 黒田裕
    • Organizer
      第4回日本生物物理学会関東支部会
    • Place of Presentation
      日本大学文理学部(東京都・世田谷区)
    • Year and Date
      2015-03-09 – 2015-03-10
    • Related Report
      2014 Research-status Report
  • [Presentation] Structural and thermodynamic study of extensively simplified BPTIs reveals a novel enthalpy stabilization mechanism through multiple alanine substitution2014

    • Author(s)
      Mohammad Monirul Islam, Keiichi Noguchi, Masafumi Yohda, Shun-ichi Kidokoro, Yutaka Kuroda
    • Organizer
      第37回日本分子生物学会
    • Place of Presentation
      パシフィコ横浜(神奈川県・横浜市)
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Research-status Report
  • [Presentation] X-ray crystallography and ELISA interaction studies reveal the sero-specificity in dengue virus2014

    • Author(s)
      Manjiri Ravindra Kulkarni, Monirul M. Islam, Montasir Elahi, Nobutaka Numoto, Nobutoshi Ito, Yutaka Kuroda
    • Organizer
      第37回日本分子生物学会
    • Place of Presentation
      パシフィコ横浜(神奈川県・横浜市)
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Research-status Report
  • [Presentation] 全原子MDシミュレーションによるアラニンスキャニング変異を導入したアミロイド形成ペプチドの凝集解析2014

    • Author(s)
      小須田 慧司, 末永 敦, 佐藤 雄士, 泰地 真弘人, 黒田 裕
    • Organizer
      第37回日本分子生物学会
    • Place of Presentation
      パシフィコ横浜(神奈川県・横浜市)
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Research-status Report
  • [Presentation] Analysis and control of amorphous protein aggregation using short peptide tags2014

    • Author(s)
      Yutaka Kuroda
    • Organizer
      JSPS Japan Hungary Joint Seminar
    • Place of Presentation
      蛋白質研究所(大阪大学)(大阪府・吹田市)
    • Year and Date
      2014-11-18 – 2014-11-20
    • Related Report
      2014 Research-status Report
    • Invited
  • [Presentation] Analysis of protein crystallization using short Solubility Controlling Peptide tags2014

    • Author(s)
      黒田裕、Mohammad M Islam
    • Organizer
      第52回日本生物物理学会年会
    • Place of Presentation
      北海道札幌コンベンションセンター(北海道・札幌市)
    • Year and Date
      2014-09-25 – 2014-09-27
    • Related Report
      2014 Research-status Report
  • [Presentation] Epitope grafted mutants provide molecular insights into sero-speci_c interactions of Dengue's ED3 with it`s monoclonal antibody(Best Poster Award;ベストポスター賞受賞)2014

    • Author(s)
      Manjiri R. Kulkarni, Montasir Elahi, Monirul M. Islam, Yutaka Kuroda
    • Organizer
      2nd UTISA Symposium-2014
    • Place of Presentation
      在日本インド大使館(東京都・千代田区)
    • Year and Date
      2014-06-27
    • Related Report
      2014 Research-status Report
  • [Presentation] 疎水性・親水性モデルを用いらないタンパク質溶解性の解析2014

    • Author(s)
      黒田 裕
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      ワークピア横浜/横浜産貿ホールマリネリア(神奈川県・横浜市)
    • Year and Date
      2014-06-25 – 2014-06-27
    • Related Report
      2014 Research-status Report
    • Invited
  • [Presentation] VanXの溶菌活性を用いた新規スクリーニング法の開発2014

    • Author(s)
      呉 楠、上岡 哲矢、黒田 裕
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      ワークピア横浜/横浜産貿ホールマリネリア(神奈川県・横浜市)
    • Year and Date
      2014-06-25 – 2014-06-27
    • Related Report
      2014 Research-status Report
  • [Presentation] Analysis of Protein Aggregation by using Dynamic Light Scattering and Static Light Scattering Techniques2014

    • Author(s)
      Md. Golam Kabir、Tamotsu Zako、Mizuo Maeda、Yutaka Kuroda
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      ワークピア横浜/横浜産貿ホールマリネリア(神奈川県・横浜市)
    • Year and Date
      2014-06-25 – 2014-06-27
    • Related Report
      2014 Research-status Report
  • [Presentation] Biophysical analysis of protein aggregation kinetics using Solubility Controlling Peptide( SCP) tags2014

    • Author(s)
      Mohammad M Islam、Alam M Khan、黒田 裕
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      ワークピア横浜/横浜産貿ホールマリネリア(神奈川県・横浜市)
    • Year and Date
      2014-06-25 – 2014-06-27
    • Related Report
      2014 Research-status Report

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Published: 2014-04-04   Modified: 2017-05-10  

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