Reconstitution of cell-free protein maturation system coupled with translation system.

• Project/Area Number: 18201040
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: The University of Tokyo
• Principal Investigator: UEDA Takuya The University of Tokyo, 大学院・新領域創成科学研究科, 教授 (80184927)
• Co-Investigator(Kenkyū-buntansha): SHIMIZU Yoshihiro 東京大学, 大学院・新領域創成科学研究科, 助教 (90401231)
• Project Period (FY): 2006 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥51,740,000 (Direct Cost: ¥39,800,000、Indirect Cost: ¥11,940,000); Fiscal Year 2008: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000); Fiscal Year 2007: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000); Fiscal Year 2006: ¥23,660,000 (Direct Cost: ¥18,200,000、Indirect Cost: ¥5,460,000)
• Keywords: 無細胞蛋白質合成系 / リボソーム / シャペロン / フォールディング / 分泌蛋白質 / 膜蛋白質 / 生体外蛋白質合成系 / フォールデング / GroEL / 翻訳後修飾 / トリッガーファクター

Research Abstract

蛋白質の成熟過程のメカニズム解明することを目標として、再構築した蛋白質合成系PURE systemによって大腸菌ゲノム上の全蛋白質(4132個)の合成を行い、各蛋白質の凝集特性を遠心分離により可溶性を評価した。PURE systemにより合成可能また電気泳動可能であった7割についての評価したところ、凝集しやすさは二峰性を示すことが示された。分子量が小さい蛋白質、等電点が低い蛋白質は可溶性が高いことが示された。また、細胞質の凝集傾向のある蛋白質792個について、シャペロン存在下のPURE systemで合成を行い、その可溶性の向上から、シャペロン依存性を評価した。以上の解析結果をe-Solデータベースとして公開した(http://tp-esol.genes.nig.ac.jp/)。

Research Products

• [Journal Article] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis (2009)
  • Author(s): Tsuboi, M., Morita, H., Nozaki, Y., Akama, K., Ueda, T.., Ito, K., Nierhaus, K. H., Takeuchi, N.
  • Journal Title: Mol Cell 35 Pages: 502-510
• [Journal Article] Real-time monitoring of cell-free translation on a quartz-crystal microbalance (2009)
  • Author(s): Takahashi, S., Iida, M., Furusawa, H., Shimizu, Y.., Ueda, T., Okahata, Y.
  • Journal Title: Journal of the American Chemical Society 131 Pages: 9326-9332
• [Journal Article] Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins (2009)
  • Author(s): Niwa, T., Ying, B. W., Saito, K., Jin, W., Takada, S., Ueda, T., Taguchi, H.
  • Journal Title: Proc Natl Acad Sci USA 106 Pages: 4201-4206
• [Journal Article] Twin-arginine-dependent translocation of Sufl in the absence of cytosolic helper proteins (2009)
  • Author(s): Holzapfel, E., Moser, M., Schiltz, E., Ueda, T.., Betton, J.M., Muller, M.
  • Journal Title: Biochemistry 48 Pages: 5096-5105
• [Journal Article] A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells (2009)
  • Author(s): Kuruma, Y., Stano, P., Ueda, T., Luisi, P .L.
  • Journal Title: Biochim Biophys Acta 1788 Pages: 567-574
• [Journal Article] Epitope mapping using ribosome display in a reconstituted cell-free protein synthesis system (2009)
  • Author(s): Osada, E., Shimizu, Y.., Akbar, B. K., Kanamori, T., Ueda, T.
  • Journal Title: J Biochem 145 Pages: 693-700
  • NAID: 10025589286
• [Journal Article] Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f^5CAU as revealed with a mitochondrial in vitro translation system (2009)
  • Author(s): Takemoto, C., Spremulli, L. L., Benkowski, L. A., Ueda, T., Yokogawa, T., Watanabe, K.
  • Journal Title: Nucleic Acids Res. 37 Pages: 1616-1627
• [Journal Article] EF-G2mt is an exclusive recycling factor in mammalian mitoch ondrial protein synthesis (2009)
  • Author(s): Tsuboi, M., Morita, H., Nozaki, Y., Akama, K., Ueda, T., Ito, K., Nierhaus, K.H., Takeuchi, N.
  • Journal Title: Mol Cell 35 Pages: 502-510
  • Peer Reviewed
• [Journal Article] Bimodal protein solubility distribution revealed by an aggre gation analysis of the entire ensemble of Escherichia coli proteins (2009)
  • Author(s): Niwa, T., Ying, B.W., Saito, K., Jin, W., Takada, S., Ueda, T., Taguchi, H.
  • Journal Title: Proc Natl Acad Sci USA 106 Pages: 4201-4206
  • Peer Reviewed
• [Journal Article] A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells (2009)
  • Author(s): Kuruma, Y., Stano, P., Ueda, T., Luisi, P.L.
  • Journal Title: Biochim Biophys Acta 1788 Pages: 567-574
  • Peer Reviewed
• [Journal Article] Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics (2008)
  • Author(s): Yamashita, R., Suzuki, Y., Takeuchi, N., Wakaguri, H., Ueda, T.., Sugano, S., Nakai, K.
  • Journal Title: Nucleic Acids Res.
• [Journal Article] Single-molecule imaging of full protein synthesis by immobilized ribosomes. (2008)
  • Author(s): Uemura, S., Iizuka, R., Ueno, T., Shimizu, Y.., Taguchi, H., Ueda, T., Puglisi, J. D., Funatsu, T.
  • Journal Title: Nucleic Acids Res.
• [Journal Article] 70 S ribosomes bind to Shine-Dalgarno sequences without required dissociations (2008)
  • Author(s): Takahashi, S., Akita, R., Matsuno, H., Furusawa, H., Shimizu, Y.., Ueda, T., Okahata, Y.
  • Journal Title: Chembiochem 9 Pages: 870-873
• [Journal Article] UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro (2008)
  • Author(s): Ozaki, Y., Suzuki, T., Kuruma, Y., Ueda, T., Yoshida, M.
  • Journal Title: Biochem Biophys Res Commun 367 Pages: 663-666
• [Journal Article] HMRF1L is a human mitochondrial translation release factor involved in the decoding of the termination codons UAA and UAG (2008)
  • Author(s): Nozaki, Y., Matsunaga, N., Ishizawa, T., Ueda, T., Takeuchi, N.
  • Journal Title: Genes Cells 13 Pages: 429-438
• [Journal Article] Polyadenylation in mammalian mitochondria : Insights from recent studies (2008)
  • Author(s): Nagaike, T., Suzuki, T., Ueda, T.
  • Journal Title: Biochim Biophys Acta 1779 Pages: 266-269
• [Journal Article] The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC (2008)
  • Author(s): Ishizawa, T., Nozaki, Y., Ueda, T.. Takeuchi, N.
  • Journal Title: Biochem Biophys Res Commun
• [Journal Article] UncI protein can mediate ring-assembly of c-subunits of FoFl-ATP synthase in vitro (2008)
  • Author(s): Ozaki, Y., Suzuki, T., Kuruma, Y., Ueda, T. and Yoshida, M
  • Journal Title: Biochem Biophys Res Commun 367 Pages: 663-666
  • Peer Reviewed
• [Journal Article] The PURE System : A Minimal Cell-Free Translation System In Kudlick, W., Katzen, F. Bennett, R. (eds.) (2007)
  • Author(s): Ying, B.-W., Shimizu, Y., Ueda, T.
  • Journal Title: Cell-Free Protein Expression. Landes Bioscience Pages: 76-83
• [Journal Article] Chaperone properties of mammaliall mitochondrial translation elongation factor Tu (2007)
  • Author(s): Suzuki, H., Ueda, T., Taguchi, H. and Takeuchi, N.
  • Journal Title: J Biol Chem 282 Pages: 4076-4084
  • Peer Reviewed
• [Journal Article] Ribosomal Protein SI Is not Essential for the trans-translation Machinery (2007)
  • Author(s): Qi, H., Shimizu, Y. and Ueda, T
  • Journal Title: J Mol Biol 368 Pages: 845-852
  • Peer Reviewed
• [Journal Article] Efficient protein selection based on ribosome display system with purified components. (2007)
  • Author(s): Ohashi, H., Shimizu, Y., Ying, B.W., Ueda, T.
  • Journal Title: Biochem Biophys Res Commun 352 Pages: 270-276
• [Journal Article] Ribosomal Protein S1 Is not Essential for the trans-translation Machinery. (2007)
  • Author(s): Qi, H., Shimizu, Y., Ueda, T.
  • Journal Title: J Mol Biol 368 Pages: 845-852
• [Journal Article] Chaperone properties of mammalian mitochondrial translation elongation factor Tu. (2007)
  • Author(s): Suzuki, H., Ueda, T., Taguchi, H., Takeuchi, N.
  • Journal Title: J Biol Chem 282 Pages: 4076-4084
• [Journal Article] Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding. (2006)
  • Author(s): Ying, B.W., Taguchi, H., Ueda, T.
  • Journal Title: J Biol Chem 281 Pages: 21813-21819
• [Journal Article] Spermidine inhibits transient and table ribosome subunit dissociation. (2006)
  • Author(s): Umekage, S., Ueda, T.
  • Journal Title: FEBS Lett 580 Pages: 1222-1226
• [Journal Article] SmpB triggers GTP hydrolysis of elongation factor Tu on Ribosome's by compensating for the lack of codon-anticodon interaction during trans-translation initiation. (2006)
  • Author(s): Shimizu, Y., Ueda, T.
  • Journal Title: J Biol Chem 281 Pages: 15987-15996
• [Journal Article] Cell-free translation systems for protein engineering. (2006)
  • Author(s): Shimizu, Y., Kuruma, Y., Ying, B.W., Umekage, S., Ueda, T.
  • Journal Title: Febs J 273 Pages: 4133-4140
• [Journal Article] Nuclear respiratory factor 2 activates transcription of human mitochondrial translation initiation factor 2 gene. (2006)
  • Author(s): Hayashi, R., Ueda, T., Farwell, M.A., Takeuchi, N.
  • Journal Title: Mitochondrion 7 Pages: 195-203
• [Presentation] 再構築型生体外蛋白質合成系を用いた大腸菌全蛋白質の凝集解析 (2009)
  • Author(s): 丹羽達也, インベイウェン, 斎藤克代, 金文珍, 高田彰二, 上田卓也, 田口英樹
  • Organizer: 日本蛋白質科学会
  • Place of Presentation: 熊本全日空ホテルニュースカイ
  • Year and Date: 2009-05-20
• [Presentation] Cell-freeを使つた膜タンパク質のリボソーム内合成 (2009)
  • Author(s): Yutetsu Kuruma, Pasquale Stano, Takuya Ueda, Pier Luigi Luisi
  • Organizer: 第3回無細胞生命科学研究会
  • Place of Presentation: 弘前大学創立50周年記念会館
  • Year and Date: 2009-03-16
• [Presentation] 無細胞蛋白質合成系における30Sリボソームサブユニットの再構成 (2009)
  • Author(s): 田丸大知, 清水義宏, 上田卓也
  • Organizer: 第3回無細胞生命科学研究会弘前大学創立50周年
  • Place of Presentation: 記念会館みちのくホール
  • Year and Date: 2009-03-16
• [Presentation] Cell-free Expression of Phospholipid-Synthesizing Membrane Proteins in the Interior of Liposomes ーリン脂質の生合成過程に関わる膜蛋白質のリボソーム内合成 (2008)
  • Author(s): Yutetsu Kuruma, Pasquale Stano, Takuya Ueda Pier Luigi Luisi
  • Organizer: 日本生物物理学会第46回年会福岡国際会議
  • Year and Date: 2008-12-03
• [Presentation] 自己複製可能な人工細胞の構築-Construction of Self-Reproducible Synthetic Cell (2008)
  • Author(s): 車命徹, Pasquale Stano, 上田卓也, Pier Luigi Luisi
  • Organizer: 「糸田胞を創る」研究会1.0
  • Place of Presentation: 大阪大学吹田キャンパス
  • Year and Date: 2008-10-16
• [Presentation] 無細胞蛋白質合成系における3OSリボソームサブユニットの再構成 (2008)
  • Author(s): 田丸大知, 清水義宏, 上田卓也
  • Organizer: 「細胞を創る」研究会1.0
  • Place of Presentation: 大阪大学吹田キャンパス銀杏会館
  • Year and Date: 2008-10-16
• [Presentation] Construction of Self-Reproducible Synthetic Cell (2008)
  • Author(s): Yutetsu Kuruma, Pasquale Stano, Takuya Ueda, Pier Luigi Luisi
  • Organizer: SYNTHETIC BIOLOGY 4.0
  • Place of Presentation: Hong kong University of Science & Technology
  • Year and Date: 2008-10-10
• [Presentation] Reconstruction of functional E. coli 30S ribosomal subunits in a cell-free system (2008)
  • Author(s): Daichi Tamaru, Yoshihiro Shimizu, Takuya Ueda
  • Organizer: The Fourth International Meeting on Synthetic Biology
  • Place of Presentation: Hong Kong University of Science and Technology
  • Year and Date: 2008-10-10
• [Presentation] 無細胞蛋白質合成系における30Sリボソームサブユニットの再構成 (2008)
  • Author(s): 田丸大知, 清水義宏, 上田卓也
  • Organizer: 第5回21世紀大腸菌研究会
  • Place of Presentation: 静岡国民年金健康センター「藤枝エミナース」
  • Year and Date: 2008-07-28
• [Presentation] 無細胞蛋白質合成系における30Sリボソームサブユニットの再構成無細胞蛋白質合成系における30Sリボソームサブユニットの再構成 (2008)
  • Author(s): 田丸大知, 清水義宏, 上田卓也
  • Organizer: 第10回日本RNA学会年会
  • Place of Presentation: 札幌市・札幌コンベンションセンター
  • Year and Date: 2008-07-23
• [Presentation] PURE system を用いた大腸菌全蛋白質 (2008)
  • Author(s): 丹羽達也, 斎藤克代, イン・ベイウェン, 金文珍, 高田彰二, 上田卓也, 田口英樹
  • Organizer: 第8回日本蛋白質科学会年会
  • Place of Presentation: タワーホール船堀 (東京)
  • Year and Date: 2008-06-11
• [Presentation] 自己複製可能な人工細胞の構築ー Construction of Self-reproducible Minimal Cell (2008)
  • Author(s): Yutetsu Kuruma, Pasquale Stano, Takuya Ueda, Pier Luigi Luisi
  • Organizer: 第8回蛋白質科学会年会
  • Place of Presentation: タワーホール船堀 (東京)
  • Year and Date: 2008-06-10
• [Presentation] 絵図・地図からみた城下町大坂 (2008)
  • Author(s): 鳴海 邦匡
  • Organizer: 大阪歴史博物館姻研究シンポジウム職下町大坂を教る」
  • Place of Presentation: 大阪府(大阪歴史博物館)
  • Year and Date: 2008-03-23
• [Presentation] PURE system による膜タンパク質の合成 (2007)
  • Author(s): 阿部真人, 金森崇, 車命徹, 西山賢一, 清水義宏, 上田卓也
  • Organizer: 日本分子生物学会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-14
• [Presentation] PURE system におけるtRNA成分の再構成 (2007)
  • Author(s): 福島伸也, 清水義宏, 大野敏, 横川隆志, 西川一八, 上田卓也
  • Organizer: 日本分子生物学会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-14
• [Presentation] 生命への constructive approach (2007)
  • Author(s): 上田卓也
  • Organizer: 日本分子生物学会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-12
• [Presentation] 蛋白質研究のために何をなすべきか (2007)
  • Author(s): 上田卓也
  • Organizer: タンパク質生産・精製技術最前線セミナー～高品質タンパク質生産と精製システムの開発を目指して～
  • Place of Presentation: 横浜ランドマークタワー
  • Year and Date: 2007-12-10
• [Presentation] 構造体としての地図一国絵図 (2007)
  • Author(s): 杉本 史子
  • Organizer: 科学研究費補助金・基盤(A)「地図史料学の構築」2007年度公開研究集会「歴史のなかの地図「地図-知の交差点」
  • Place of Presentation: 東京都(東京大学)
  • Year and Date: 2007-07-08
• [Presentation] コンパスにみる近世日本の測量術の画期 (2007)
  • Author(s): 鳴海 邦匡
  • Organizer: 科学研究費補助金・基盤(A)「地図史料学の構築」2007年度公開研究集会「歴史のなかの地図[地図-知の交差点」
  • Place of Presentation: 東京都(東京大学)
  • Year and Date: 2007-07-07
• [Presentation] 認知の道具としての地図 (2007)
  • Author(s): 中村 雄祐
  • Organizer: 科学研究費補助金・基盤(A)「地図史料学の構築」2007年度公開研究集会「歴史のなかの地図「地図-知の交差点」
  • Place of Presentation: 東京都(東京大学)
  • Year and Date: 2007-07-07
• [Presentation] 近世日本のアーカイブズー利用の側面を中心に- (2007)
  • Author(s): 冨善 一敏
  • Organizer: 日本アーカイプズ学会2007年度大会企画研究会「アーカイブズの〈力〉-歴史からの検証-
  • Place of Presentation: 学習院大学
  • Year and Date: 2007-04-22
• [Book] The PURE System:A Minimal Cell-Free Translation System. In Kudlick, W., Katzen, F. and Bennett, R.(eds.), Cell-Free Protein Expression (2007)
  • Author(s): Ying, B.W., Shimizu, Y. and Ueda, T.
  • Publisher: (2007).Landes Bioscience,Austin
• [Remarks]
  • URL: http://tp-esol.genes.nig.ac.jp/
• [Remarks]
  • URL: http://molbio.t.u-tokyo.ac.jp/publication.html
• [Remarks]
  • URL: http://molbio.t.u-tokyo.ac.jp/publication.html