詳細検索はこちら

澤崎 達也サワサキ タツヤ

所属部署
プロテオサイエンスセンター
職名教授
メールアドレスsawasaki.tatsuya.mf[at]ehime-u.ac.jp ※[at]を@に書き換えて送信して下さい
ホームページURLhttp://www.pros.ehime-u.ac.jp/cell-free/
生年月日
Last Updated :2017/08/18

研究者基本情報

学歴

  • 1992年04月 - 1998年09月, 広島大学大学院, 理学研究科, 生物科学専攻
  • 1988年04月 - 1992年03月, 広島大学, 理学部, 生物学科植物学専攻

経歴

  •   2013年04月,  - 現在, 愛媛大学, プロテオサイエンスセンター, 教授
  •   2012年04月,  - 2013年03月, 愛媛大学, 無細胞生命科学工学研究センター, 教授
  •   2003年04月,  - 2012年03月, 愛媛大学, 無細胞生命科学工学研究センター, 准教授
  •   1999年04月,  - 2003年03月, 愛媛大学, 工学部応用化学科, 助教
  •   1998年10月,  - 1999年03月, 愛媛大学, 工学部応用化学科, ポスドク

所属学協会

  • 日本生化学学会
  • 日本分子生物学会
  • American Society of Cell Biology

研究活動情報

研究分野

  • システムゲノム科学
  • 機能生物化学
  • 植物分子生物・生理学
  • 腫瘍生物学
  • 生物機能・バイオプロセス
  • 構造生物化学
  • 病態検査学

研究キーワード

    コムギ胚芽, プロテインアレイ, 無細胞タンパク質合成, 細胞死, 細胞内シグナル, 蛋白質代謝, タンパク質, 無細胞蛋白質合成系, プロテインカイネース, カスパーゼ, プロテアーゼ, 切断, Alpha Screen, カスパーゼ3, プロテオリシス, ユビキチン, ユビキチン化, RING, がん抑制タンパク質, 蛋白ライブラリー, 炎症, 蛋白質ネットワーク, E3リガーゼ, 無脂肪タンパク質合成, 蛋白質ライブラリー, p53, ポリユビキチン鎖, シグナル伝達, 翻訳, セレノメチオニン標識, 無細胞, 無細胞蛋白質合成

論文

  • MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis.
    Santolini M, Sakakibara I, Gauthier M, Ribas-Aulinas F, Takahashi H, Sawasaki T, Mouly V, Concordet JP, Defossez PA, Hakim V, Maire P, Nucleic acids research,   2016年06月
  • Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.
    Shi G, Ando T, Suzuki R, Matsuda M, Nakashima K, Ito M, Omatsu T, Oba M, Ochiai H, Kato T, Mizutani T, Sawasaki T, Wakita T, Suzuki T, PLoS pathogens, 12, (2) ,   2016年02月
  • Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells.
    Suzuki J, Maruyama S, Tamauchi H, Kuwahara M, Horiuchi M, Mizuki M, Ochi M, Sawasaki T, Zhu J, Yasukawa M, Yamashita M, Immunology, 147, (4) 476 - 487,   2016年04月
  • Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.
    Suzuki Y, Chin WX, Han Q, Ichiyama K, Lee CH, Eyo ZW, Ebina H, Takahashi H, Takahashi C, Tan BH, Hishiki T, Ohba K, Matsuyama T, Koyanagi Y, Tan YJ, Sawasaki T, Chu JJ, Vasudevan SG, Sano K, Yamamoto N, PLoS pathogens, 12, (1) ,   2016年01月
  • Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.
    Takahashi H, Uematsu A, Yamanaka S, Imamura M, Nakajima T, Doi K, Yasuoka S, Takahashi C, Takeda H, Sawasaki T, PloS one, 11, (6) ,   2016年
  • AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.
    Yano T, Takeda H, Uematsu A, Yamanaka S, Nomura S, Nemoto K, Iwasaki T, Takahashi H, Sawasaki T, PloS one, 11, (6) ,   2016年
  • Reconstituted AIM2 inflammasome in cell-free system.
    Kaneko N, Ito Y, Iwasaki T, Takeda H, Sawasaki T, Migita K, Agematsu K, Kawakami A, Morikawa S, Mokuda S, Kurata M, Masumoto J, Journal of immunological methods, 426, 76 - 81,   2015年11月
  • Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.
    Ramadan A, Nemoto K, Seki M, Shinozaki K, Takeda H, Takahashi H, Sawasaki T, BMC plant biology, 15,   2015年11月
  • Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model.
    Nakajima M, Nagase S, Iida M, Takeda S, Yamashita M, Watari A, Shirasago Y, Fukasawa M, Takeda H, Sawasaki T, Yagi K, Kondoh M, The Journal of pharmacology and experimental therapeutics, 354, (3) 440 - 447,   2015年09月
  • Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly.
    Ohnishi T, Yanazawa M, Sasahara T, Kitamura Y, Hiroaki H, Fukazawa Y, Kii I, Nishiyama T, Kakita A, Takeda H, Takeuchi A, Arai Y, Ito A, Komura H, Hirao H, Satomura K, Inoue M, Muramatsu S, Matsui K, Tada M, Sato M, Saijo E, Shigemitsu Y, Sakai S, Umetsu Y, Goda N, Takino N, Takahashi H, Hagiwara M, Sawasaki T, Iwasaki G, Nakamura Y, Nabeshima Y, Teplow DB, Hoshi M, Proceedings of the National Academy of Sciences of the United States of America, 112, (32) E4465 - 74,   2015年08月
  • Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues.
    Nemoto K, Takemori N, Seki M, Shinozaki K, Sawasaki T, The Journal of biological chemistry, 290, (27) 16665 - 16677,   2015年07月
  • Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.
    Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T, Endo Y, Sawasaki T, Scientific reports, 5,   2015年06月
  • High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.
    Takemori N, Takemori A, Matsuoka K, Morishita R, Matsushita N, Aoshima M, Takeda H, Sawasaki T, Endo Y, Higashiyama S, Molecular bioSystems, 11, (2) 361 - 365,   2015年02月
  • Novel Autoantigens Associated with Lupus Nephritis.
    Onishi S, Adnan E, Ishizaki J, Miyazaki T, Tanaka Y, Matsumoto T, Suemori K, Shudou M, Okura T, Takeda H, Sawasaki T, Yasukawa M, Hasegawa H, PloS one, 10, (6) ,   2015年
  • Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.
    Kinoshita-Kikuta E, Kinoshita E, Eguchi Y, Yanagihara S, Edahiro K, Inoue Y, Taniguchi M, Yoshida M, Yamamoto K, Takahashi H, Sawasaki T, Utsumi R, Koike T, PloS one, 10, (7) ,   2015年
  • Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD.
    Furukawa H, Oka S, Shimada K, Masuo K, Nakajima F, Funano S, Tanaka Y, Komiya A, Fukui N, Sawasaki T, Tadokoro K, Nose M, Tsuchiya N, Tohma S, Biomarker insights, 10, 63 - 73,   2015年
  • A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.
    Matsunaga S, Masaoka T, Sawasaki T, Morishita R, Iwatani Y, Tatsumi M, Endo Y, Yamamoto N, Sugiura W, Ryo A, Frontiers in microbiology, 6,   2015年
  • The apoptotic initiator caspase-8: its functional ubiquity and genetic diversity during animal evolution.
    Sakamaki K, Shimizu K, Iwata H, Imai K, Satou Y, Funayama N, Nozaki M, Yajima M, Nishimura O, Higuchi M, Chiba K, Yoshimoto M, Kimura H, Gracey AY, Shimizu T, Tomii K, Gotoh O, Akasaka K, Sawasaki T, Miller DJ, Molecular biology and evolution, 31, (12) 3282 - 3301,   2014年12月
  • The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes.
    Arimitsu E, Ogasawara T, Takeda H, Sawasaki T, Ikeda Y, Hiasa Y, Maeyama K, European journal of pharmacology, 745, 117 - 122,   2014年12月
  • ScreenCap3: Improving prediction of caspase-3 cleavage sites using experimentally verified noncleavage sites.
    Fu SC, Imai K, Sawasaki T, Tomii K, Proteomics, 14, (17-18) 2042 - 2046,   2014年09月
  • Identification of RFPL3 protein as a novel E3 ubiquitin ligase modulating the integration activity of human immunodeficiency virus, type 1 preintegration complex using a microtiter plate-based assay.
    Tan BH, Suzuki Y, Takahashi H, Ying PH, Takahashi C, Han Q, Chin WX, Chao SH, Sawasaki T, Yamamoto N, Suzuki Y, The Journal of biological chemistry, 289, (38) 26368 - 26382,   2014年09月
  • Pctaire1/Cdk16 promotes skeletal myogenesis by inducing myoblast migration and fusion.
    Shimizu K, Uematsu A, Imai Y, Sawasaki T, FEBS letters, 588, (17) 3030 - 3037,   2014年08月
  • Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.
    Masaki T, Matsunaga S, Takahashi H, Nakashima K, Kimura Y, Ito M, Matsuda M, Murayama A, Kato T, Hirano H, Endo Y, Lemon SM, Wakita T, Sawasaki T, Suzuki T, Journal of virology, 88, (13) 7541 - 7555,   2014年07月
  • Profiling of autoantibodies in sera of pancreatic cancer patients.
    Nagayoshi Y, Nakamura M, Matsuoka K, Ohtsuka T, Mori Y, Kono H, Aso T, Ideno N, Takahata S, Ryo A, Takeda H, Ito T, Oda Y, Endo Y, Sawasaki T, Tanaka M, Annals of surgical oncology, 21 Suppl 3, S459 - 65,   2014年06月
  • Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.
    Takiuchi T, Nakagawa T, Tamiya H, Fujita H, Sasaki Y, Saeki Y, Takeda H, Sawasaki T, Buchberger A, Kimura T, Iwai K, Genes to cells : devoted to molecular & cellular mechanisms, 19, (3) 254 - 272,   2014年03月
  • The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions.
    Kudoh A, Takahama S, Sawasaki T, Ode H, Yokoyama M, Okayama A, Ishikawa A, Miyakawa K, Matsunaga S, Kimura H, Sugiura W, Sato H, Hirano H, Ohno S, Yamamoto N, Ryo A, Retrovirology, 11,   2014年01月
  • Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease in Nicotiana benthamiana.
    Ito M, Takahashi H, Sawasaki T, Ohnishi K, Hikichi Y, Kiba A, Plant signaling & behavior, 9, (1) ,   2014年
  • Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.
    Mitsunami T, Nishihara M, Galis I, Alamgir KM, Hojo Y, Fujita K, Sasaki N, Nemoto K, Sawasaki T, Arimura G, PloS one, 9, (9) ,   2014年
  • Anti-interleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels.
    Ishigami T, Abe K, Aoki I, Minegishi S, Ryo A, Matsunaga S, Matsuoka K, Takeda H, Sawasaki T, Umemura S, Endo Y, FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27, (9) 3437 - 3445,   2013年09月
  • The Solanum chacoense ovary receptor kinase 11 (ScORK11) undergoes tissue-dependent transcriptional, translational and post-translational regulation.
    Germain H, Gray-Mitsumune M, Houde J, Benhamman R, Sawasaki T, Endo Y, Matton DP, Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale, 70, 261 - 268,   2013年09月
  • Nek5, a novel substrate for caspase-3, promotes skeletal muscle differentiation by up-regulating caspase activity.
    Shimizu K, Sawasaki T, FEBS letters, 587, (14) 2219 - 2225,   2013年07月
  • [Cell-free based protein array technology for analyses of protein kinases and ubiquitin ligases].
    Sawasaki T, Takeda H, Takahashi H, Nemoto K, Seikagaku. The Journal of Japanese Biochemical Society, 85, (6) 438 - 446,   2013年06月
  • Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.
    Iwasaki T, Katayama T, Kohama K, Endo Y, Sawasaki T, Molecular biology of the cell, 24, (6) 748 - 756,   2013年03月
  • Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method.
    Mizutani Y, Matsuoka K, Takeda H, Shiogama K, Inada K, Hayakawa K, Yamada H, Miyazaki T, Sawasaki T, Endo Y, Tsutsumi Y, Journal of immunological methods, 387, (1-2) 57 - 70,   2013年01月
  • Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.
    Nakano M, Nishihara M, Yoshioka H, Takahashi H, Sawasaki T, Ohnishi K, Hikichi Y, Kiba A, PloS one, 8, (9) ,   2013年
  • Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.
    Takahashi H, Takahashi C, Moreland NJ, Chang YT, Sawasaki T, Ryo A, Vasudevan SG, Suzuki Y, Yamamoto N, Antiviral research, 96, (3) 305 - 314,   2012年12月
  • The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model.
    Kominami K, Nakabayashi J, Nagai T, Tsujimura Y, Chiba K, Kimura H, Miyawaki A, Sawasaki T, Yokota H, Manabe N, Sakamaki K, Biochimica et biophysica acta, 1823, (10) 1825 - 1840,   2012年10月
  • Interferon-induced SCYL2 limits release of HIV-1 by triggering PP2A-mediated dephosphorylation of the viral protein Vpu.
    Miyakawa K, Sawasaki T, Matsunaga S, Tokarev A, Quinn G, Kimura H, Nomaguchi M, Adachi A, Yamamoto N, Guatelli J, Ryo A, Science signaling, 5, (245) ,   2012年10月
  • Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system.
    Takahashi H, Ozawa A, Nemoto K, Nozawa A, Seki M, Shinozaki K, Takeda H, Endo Y, Sawasaki T, FEBS letters, 586, (19) 3134 - 3141,   2012年09月
  • Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis.
    Matsunaga S, Sawasaki T, Ode H, Morishita R, Furukawa A, Sakuma R, Sugiura W, Sato H, Katahira M, Takaori-Kondo A, Yamamoto N, Ryo A, Journal of proteomics, 75, (15) 4863 - 4873,   2012年08月
  • A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity.
    Kiba A, Nakano M, Vincent-Pope P, Takahashi H, Sawasaki T, Endo Y, Ohnishi K, Yoshioka H, Hikichi Y, Journal of plant physiology, 169, (10) 1017 - 1022,   2012年07月
  • Stress-inducible caspase substrate TRB3 promotes nuclear translocation of procaspase-3.
    Shimizu K, Takahama S, Endo Y, Sawasaki T, PloS one, 7, (8) ,   2012年
  • In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis.
    Kominami K, Nagai T, Sawasaki T, Tsujimura Y, Yashima K, Sunaga Y, Tsuchimochi M, Nishimura J, Chiba K, Nakabayashi J, Koyamada K, Endo Y, Yokota H, Miyawaki A, Manabe N, Sakamaki K, PloS one, 7, (11) ,   2012年
  • Autophosphorylation profiling of Arabidopsis protein kinases using the cell-free system.
    Nemoto K, Seto T, Takahashi H, Nozawa A, Seki M, Shinozaki K, Endo Y, Sawasaki T, Phytochemistry, 72, (10) 1136 - 1144,   2011年07月
  • Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.
    Tsuge S, Mizutani Y, Matsuoka K, Sawasaki T, Endo Y, Naruishi K, Maeda H, Takashiba S, Shiogama K, Inada K, Tsutsumi Y, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 59, (7) 673 - 689,   2011年07月
  • Caspase-8 cleavage of the interleukin-21 (IL-21) receptor is a negative feedback regulator of IL-21 signaling.
    Akagi T, Shimizu K, Takahama S, Iwasaki T, Sakamaki K, Endo Y, Sawasaki T, FEBS letters, 585, (12) 1835 - 1840,   2011年06月
  • Wheat germ cell-free protein production system for post-genomic research.
    Madono M, Sawasaki T, Morishita R, Endo Y, New biotechnology, 28, (3) 211 - 217,   2011年04月
  • Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.
    Nozawa A, Ogasawara T, Matsunaga S, Iwasaki T, Sawasaki T, Endo Y, BMC biotechnology, 11,   2011年04月
  • Cell-free-based protein microarray technology using agarose/DNA microplate.
    Sawasaki T, Endo Y, Methods in molecular biology (Clifton, N.J.), 607, 63 - 72,   2010年
  • An efficient approach to the production of vaccines against the malaria parasite.
    Tsuboi T, Takeo S, Sawasaki T, Torii M, Endo Y, Methods in molecular biology (Clifton, N.J.), 607, 73 - 83,   2010年
  • Cell-free protein synthesis for structure determination by X-ray crystallography.
    Watanabe M, Miyazono K, Tanokura M, Sawasaki T, Endo Y, Kobayashi I, Methods in molecular biology (Clifton, N.J.), 607, 149 - 160,   2010年
  • [Membrane protein production using wheat germ cell-free system].
    Nozawa A, Tozawa Y, Sawasaki T, Endo Y, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 54, (12 Suppl) 1443 - 1447,   2009年09月
  • Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag.
    Nishi M, Ryo A, Tsurutani N, Ohba K, Sawasaki T, Morishita R, Perrem K, Aoki I, Morikawa Y, Yamamoto N, FEBS letters, 583, (8) 1243 - 1250,   2009年04月
  • Isolation and identification of ubiquitin-related proteins from Arabidopsis seedlings.
    Igawa T, Fujiwara M, Takahashi H, Sawasaki T, Endo Y, Seki M, Shinozaki K, Fukao Y, Yanagawa Y, Journal of experimental botany, 60, (11) 3067 - 3073,   2009年
  • DNA-binding profiling of human hormone nuclear receptors via fluorescence correlation spectroscopy in a cell-free system.
    Kobayashi T, Kodani Y, Nozawa A, Endo Y, Sawasaki T, FEBS letters, 582, (18) 2737 - 2744,   2008年08月
  • RIP and RALyase cleave the sarcin/ricin domain, a critical domain for ribosome function, during senescence of wheat coleoptiles.
    Sawasaki T, Nishihara M, Endo Y, Biochemical and biophysical research communications, 370, (4) 561 - 565,   2008年06月
  • The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta.
    Masaoka T, Nishi M, Ryo A, Endo Y, Sawasaki T, FEBS letters, 582, (13) 1795 - 1801,   2008年06月
  • A set of ligation-independent in vitro translation vectors for eukaryotic protein production.
    Bardóczy V, Géczi V, Sawasaki T, Endo Y, Mészáros T, BMC biotechnology, 8,   2008年03月
  • SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag.
    Ryo A, Tsurutani N, Ohba K, Kimura R, Komano J, Nishi M, Soeda H, Hattori S, Perrem K, Yamamoto M, Chiba J, Mimaya J, Yoshimura K, Matsushita S, Honda M, Yoshimura A, Sawasaki T, Aoki I, Morikawa Y, Yamamoto N, Proceedings of the National Academy of Sciences of the United States of America, 105, (1) 294 - 299,   2008年01月
  • Chapter 2. Development of key technologies for high-throughput cell-free protein production with the extract from wheat embryos.
    Takai K, Sawasaki T, Endo Y, Advances in protein chemistry and structural biology, 75, 53 - 84,   2008年
  • Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense.
    Germain H, Houde J, Gray-Mitsumune M, Sawasaki T, Endo Y, Rivoal J, Matton DP, FEBS letters, 581, (26) 5137 - 5142,   2007年10月
  • Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.
    Abe M, Ohno S, Yokogawa T, Nakanishi T, Arisaka F, Hosoya T, Hiramatsu T, Suzuki M, Ogasawara T, Sawasaki T, Nishikawa K, Kitamura M, Hori H, Endo Y, Proteins, 67, (3) 643 - 652,   2007年05月
  • Construction of intramolecular luciferase complementation probe for detecting specific RNA.
    Endoh T, Mie M, Funabashi H, Sawasaki T, Endo Y, Kobatake E, Bioconjugate chemistry, 18, (3) 956 - 962,   2007年05月
  • [Wheat germ cell-free protein synthesis].
    Endo Y, Sawasaki T, Seikagaku. The Journal of Japanese Biochemical Society, 79, (3) 229 - 238,   2007年03月
  • [Development of a high-throughput biochemical annotation method of genetic information based on cell-free protein synthesis, and its application to protein biology].
    Sawasaki T, Endo Y, Seikagaku. The Journal of Japanese Biochemical Society, 79, (3) 278 - 286,   2007年03月
  • [Exploration of collagen disease-related proteins based on pathogenomics: application of a cell-free protein synthesis system ].
    Nose M, Komori H, Miyazaki T, Sawasaki T, Endo Y, Seikagaku. The Journal of Japanese Biochemical Society, 79, (3) 287 - 295,   2007年03月
  • Novel protein fold discovered in the PabI family of restriction enzymes.
    Miyazono K, Watanabe M, Kosinski J, Ishikawa K, Kamo M, Sawasaki T, Nagata K, Bujnicki JM, Endo Y, Tanokura M, Kobayashi I, Nucleic acids research, 35, (6) 1908 - 1918,   2007年
  • Methods for high-throughput materialization of genetic information based on wheat germ cell-free expression system.
    Sawasaki T, Morishita R, Gouda MD, Endo Y, Methods in molecular biology (Clifton, N.J.), 375, 95 - 106,   2007年
  • The wheat germ cell-free expression system: methods for high-throughput materialization of genetic information.
    Sawasaki T, Gouda MD, Kawasaki T, Tsuboi T, Tozawa Y, Takai K, Endo Y, Methods in molecular biology (Clifton, N.J.), 310, 131 - 144,   2005年
  • Advances in genome-wide protein expression using the wheat germ cell-free system.
    Endo Y, Sawasaki T, Methods in molecular biology (Clifton, N.J.), 310, 145 - 167,   2005年
  • Purification and sequence determination of an RNA ligase from wheat embryos.
    Makino S, Sawasaki T, Endo Y, Takai K, Nucleic acids symposium series (2004), (49) 319 - 320,   2005年
  • Detection of protein-DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy.
    Kobayashi T, Okamoto N, Sawasaki T, Endo Y, Analytical biochemistry, 332, (1) 58 - 66,   2004年09月
  • [In vitro protein synthesis system: cell-free protein synthesis system prepared from wheat germ].
    Sawasaki T, Endo Y, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 49, (11 Suppl) 1514 - 1519,   2004年08月
  • Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system.
    Sawasaki T, Hasegawa Y, Morishita R, Seki M, Shinozaki K, Endo Y, Phytochemistry, 65, (11) 1549 - 1555,   2004年06月
  • Formation of circular polyribosomes in wheat germ cell-free protein synthesis system.
    Madin K, Sawasaki T, Kamura N, Takai K, Ogasawara T, Yazaki K, Takei T, Miura K, Endo Y, FEBS letters, 562, (1-3) 155 - 159,   2004年03月
  • High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.
    Endo Y, Sawasaki T, Journal of structural and functional genomics, 5, (1-2) 45 - 57,   2004年
  • Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ.
    Kawasaki T, Gouda MD, Sawasaki T, Takai K, Endo Y, European journal of biochemistry / FEBS, 270, (23) 4780 - 4786,   2003年12月
  • Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.
    Miyamoto-Sato E, Takashima H, Fuse S, Sue K, Ishizaka M, Tateyama S, Horisawa K, Sawasaki T, Endo Y, Yanagawa H, Nucleic acids research, 31, (15) ,   2003年08月
  • [Recent advances in cell-free protein synthesis: application for postgenome sciences].
    Endo Y, Sawasaki T, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 48, (4 Suppl) 549 - 554,   2003年03月
  • OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase.
    Ito Y, Ozawa A, Sawasaki T, Endo Y, Ochi K, Tozawa Y, Bioscience, biotechnology, and biochemistry, 66, (12) 2727 - 2731,   2002年12月
  • [High-throughput expression of proteins from cDNAs catalogue from Arabidopsis in wheat germ cell-free protein synthesis system].
    Sawasaki T, Seki M, Sinozaki K, Endo Y, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 47, (8 Suppl) 1003 - 1008,   2002年06月
  • [Highly efficient cell-free protein synthesis system prepared from wheat embryos].
    Endo Y, Sawasaki T, Seikagaku. The Journal of Japanese Biochemical Society, 74, (4) 326 - 330,   2002年04月
  • Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.
    Doi N, Takashima H, Kinjo M, Sakata K, Kawahashi Y, Oishi Y, Oyama R, Miyamoto-Sato E, Sawasaki T, Endo Y, Yanagawa H, Genome research, 12, (3) 487 - 492,   2002年03月

MISC

  • In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: The application of the enzyme-labeled antigen method
    Y. Mizutani, S. Tsuge, S. Tsuge, H. Takeda, Y. Hasegawa, K. Shiogama, T. Onouchi, K. Inada, T. Sawasaki, Y. Tsutsumi, Molecular Oral Microbiology, 29,   2014年01月01日, Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis. © 2014 The Authors.
  • RNA N-glycosidase activity of ribosome-inactivating proteins
    Kazuyuki Takai, Tatsuya Sawasaki, Yaeta Endo, Plant Cell Monographs, 18,   2010年12月01日, Mammalian and bacterial ribosomes have ribosomal RNAs comprising 7,000 and 5,000 nucleotides, respectively. The RNA N-glycosidase activity of ricin and other ribosome-inactivating proteins (RIPs) specifically catalyzes removal of single adenine in the sarcin/ricin loop of the largest (28S or 23S) rRNA. Breakage of this single N-glycosidic bond is entirely responsible for the cytotoxicity. Ricin recognizes a highly ordered three-dimensional structure of the sarcin/ricin domain, which directly interacts with elongation factors to help switching through different states of the ribosome during the translation elongation cycle. Plants have an enzyme that specifically cleaves the phophodiester bond at the depurinated ricin site of 28S rRNA, named ribosomal RNA apurinic site-specific lyase (RALyase). The set of RIP and RALyase and the depurination and cleavage of the 28S rRNA are likely to have a role in senescence in plants. © Springer-Verlag Berlin Heidelberg 2010.
  • RALyase; a terminator of elongation function of depurinated ribosomes
    OZAWA A, SAWASAKI T, TAKAI K, UCHIUMI T, HORI H, ENDO Y, FEBS Letts, 555, (3) 455 - 458,   2003年12月18日, Plant ribosomal RNA apurinic site specific lyase (RALyase) cleaves the phosphodiester bond at the depurinated site produced by ribosome-inactivating protein, while the biological role of this enzyme is not clear. As the depurinated ribosomes retain weak translation elongation activities, it was suggested that RALyase completes the ribosome inactivation. To confirm this point, we measured the effects of the phosphodiester cleavage using a fusion of wheat RALyase produced with a cell-free protein synthesis system from wheat germ. The results indicated that RALyase diminishes the residual elongation activities of the depurinated ribosomes. © 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • The Wheat Germ Cell-Free Protein Synthesis System
    Tatsuya Sawasaki, Yaeta Endo, Cell-Free Protein Synthesis: Methods and Protocols,   2008年07月14日
  • Cell-Free Expression System for Eukaryotic Proteins
    Yaeta Endo, Tatsuya Sawasaki, Plant Proteomics: Technologies, Strategies, and Applications,   2008年01月07日
  • Methods for high-throughput materialization of genetic information based on wheat germ cell-free expression system
    Tatsuya Sawasaki, Ryo Morishita, Mudeppa D. Gouda, Yaeta Endo, Methods in Molecular Biology, 375,   2007年04月04日, Among the cell-free protein synthesis systems, the wheat germ-based translation system has significant advantages for the high-throughput production of eukaryotic multidomain proteins in folded state. Here, we describe protocols for this cell-free expression system. © Humana Press Inc.
  • A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos; Plants apparently contain a suicide system directed at ribosomes
    MADIN K, SAWASAKI T, OGASAWARA T, ENDO Y, Proceedings of the National Academy of Science of the United States of America, 97, (2) 559 - 564,   2000年
  • Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity: tRNA recognition mechanism of the enzyme
    HORI H, KUBOTA S, WATANABE K, KIM J‐M, OGASAWARA T, SAWASAKI T, ENDO Y, J. Biol. Chem., 278, (27) 25081 - 25090,   2003年
  • Stable transformation of Arabidopsis with the bar gene using particle bombardment.
    SAWASAKI T, SEKI M, ANZAI H, IRIFUNE K, MORIKAWA H, Transgenic Research, 3, (5) 279 - 286,   1994年
  • Transgenic "Air-pollutant-philic plants" produced by particle bombardment(共著)
    Research in Photosynthesis, Klumer Academic Publishers., IV,   1992年
  • Ribonuclease Activity of Rat Liver Perchloric-Acid-soluble Protein, a Potent Inhibitor of Protein Synthesis
    MORISHITA R, KAWAGOSHI A, SAWASAKI T, MADIN K, OGASAWARA T, OKA T, ENDO Y, The Journal of Bioclogical Chemistry, 274, (29) 20688 - 20692,   1999年
  • High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system
    ENDO Y, SAWASAKI T, Biotechnology Advances, 21, (8) 695 - 713,   2003年11月01日, Cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we review our recent advances in a cell-free protein synthesis system prepared from wheat embryos. We first addressed and resolved the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins. We found that conventional wheat germ extracts contained the RNA N-glycosidase tritin and other inhibitors such as thionin, ribonucleases, deoxyribonucleases, and proteases that originate from the endosperm and inhibit translation. Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. To maximize the translation yield and throughput of the system, we then focused on developing the following issues: optimization of the ORF flanking regions, a new strategy to construct PCR-generated DNAs for screening, and design of an expression vector for large-scale protein production. The resulting system achieves high-throughput expression, with a PCR-directed system at least 50 genes that can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Under the dialysis mode of reaction, the system with the expression vector can maintain productive translation for 14 days. The cell-free system described here bypasses most of the biological processes and lends itself to robotic automation for high-throughput expression of genetic information, thus opening up many possibilities in the post-genome era. © 2003 Elsevier Inc. All rights reserved.
  • A new class of enzyme acting on damaged ribosomes : ribosomal RNA apurinic site specific lyase found in wheat germ
    OGASAWARA T, SAWASAKI T, MORISHITA R, OZAWA A, MADIN K, ENDO Y, The EMBO Journal, 18, (22) 6522 - 6531,   1999年
  • Application of cell-free translation systems to studies of cofactor binding proteins
    Masato Abe, Hiroyuki Hori, Takeshi Nakanishi, Fumio Arisaka, Tomio Ogasawara, Tatsuya Sawasaki, Masaya Kitamura, Yaeta Endo, Nucleic Acids Res. Symp. Ser., 48,   2004年12月01日, To develop applications of in vitro cell-free translation systems for production and characterization of cofactor binding proteins, we investigate the production of apo- or holo-forms of Flavin Mono Nucleotide (FMN)-binding protein from Desulfovibrio vulgaris (Miyazaki F) and purified them. The redox potential analysis and measurements of UV-, visible, and fluorescent spectra of reconstructed holo-protein showed that the FMN correctly bound to the FMN binding site. On the other hand, contrary to our expectation, we found that the apo-protein formed a dimer structure and the incorporation of the FMN led the conformational alterations of the protein. These studies demonstrate the utility of cell-free translation systems to analyses of cofactor-binding proteins.
  • Structures of transgcne loci in transgenic Arobidopsis plants obtaincd by porticle bombardment : Jvnction vegions can bind to nudear matrices
    Gcne, 218, (1) 27 - 35,   1998年
  • Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice.
    MIYAZAKI Tatsuhiko, ONO Masao, QU Wei‐Min, ZHANG Ming‐Cai, MORI Shiro, NAKATSURU Shuichi, NAKAMURA Yusuke, SAWASAKI Tatsuya, ENDO Yaeta, WeilyEuropean Journal of Immunology, 35, (5) 1510 - 1520,   2005年05月01日, Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp-Faslpr/lpr (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ-Faslpr/lpr (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr × C3H/lpr)F2 mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score = 4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method "cell-free system" with wheat germ ribosomes, the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-α, IL-1β and IFN-γ in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • A bilayer cell-free protein synthesis system for high-throughput screening of gene products
    SAWASAKI T, HASEGAWA Y, TSUCHIMOCHI M, KAMURA N, OGASAWARA T, KUROITA T, ENDO Y, Federation of European Biochemical Societies Letters, 514, (1) 102 - 105,   2002年
  • A cell-free protein synthesis system for high-throughput proteomics
    SAWASAKI T, OGASAWARA T, MORISHITA R, ENDO Y, Proceedings of the National Academy of Science of the United States of America, 99, (23) 14652 - 14657,   2002年
  • Selection of 5'-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis from wheat embryos
    KAMURA Nami, KAMURA Nami, KAMURA Nami, SAWASAKI Tatsuya, SAWASAKI Tatsuya, SAWASAKI Tatsuya, KASAHARA Yuko, TAKAI Kazuyuki, TAKAI Kazuyuki, TAKAI Kazuyuki, ENDO Yaeta, ENDO Yaeta, ENDO Yaeta, Bioorganic & Medicinal Chemistry Letters, 15, (24) 5402 - 5406,   2005年12月15日, Random libraries of mRNA 5′-leader sequences were screened to obtain some sequences that can stimulate the translation initiation in a cell-free translation system from wheat embryos as efficiently as the Ω sequence from tobacco mosaic virus. Several sequences that are as useful as the Ω sequence and are homologous to no known sequences survived the screening. We expect that these sequences add useful options to the cell-free protein synthesis system that is becoming a powerful tool in the post-genomic researches. © 2005 Elsevier Ltd. All rights reserved.
  • Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: limited inter-domain misfolding in a eukaryotic translation system
    Nobutaka Hirano, Nobutaka Hirano, Tatsuya Sawasaki, Tatsuya Sawasaki, Yuzuru Tozawa, Yuzuru Tozawa, Yaeta Endo, Yaeta Endo, Yaeta Endo, Kazuyuki Takai, Kazuyuki Takai, Kazuyuki Takai, Kazuyuki Takai, Kazuyuki Takai, Proteins: Structure, Function, and Bioinformatics, 64, (2) 343 - 354,   2006年08月01日, It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of β-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains. © 2006 Wiley-Liss, Inc.
  • Activity-based in vitro selection of T4 DNA ligase
    TAKAHASHI Fumio, TAKAHASHI Fumio, FUNABASHI Hisakage, MIE Masayasu, ENDO Yaeta, SAWASAKI Tatsuya, AIZAWA Masuo, KOBATAKE Eiry, ElsevierBiochemical and Biophysical Research Communications, 336, (3) 987 - 993,   2005年10月28日, Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3′ end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA. © 2005 Elsevier Inc. All rights reserved.
  • Formation of circular polysomes in wheat germ cell-free protein synthesis system
    Elsevier ScienceFEBS Letters, 555, (3) 455 - 458,   2004年
  • Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract.
    MAKINO Shin−ichi, MAKINO Shin−ichi, SAWASAKI Tatsuya, SAWASAKI Tatsuya, TOZAWA Yuzuru, ENDO Yaeta, ENDO Yaeta, ENDO Yaeta, TAKAI Kazuyuki, TAKAI Kazuyuki, TAKAI Kazuyuki, Biochemical and Biophysical Research Communications, 347, (4) 1080 - 1087,   2006年09月08日, Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed. © 2006 Elsevier Inc. All rights reserved.
  • The initiator caspase, caspase-10β, and the BH-3-only molecule, Bid, demonstrate evolutionary conservation in Xenopus of their pro-apoptotic activities in the extrinsic and intrinsic pathways
    KOMINAMI Katsuya, TAKAGI Chiyo, KURATA Tomoko, KITAYAMA Atsushi, NOZAKI Masami, SAWASAKI Tatsuya, KUIDA Keisuke, ENDO Yaeta, MANABE Noboru, UENO Naoto, SAKAMAKI Kazuhiro, Genes to Cells, 11, (7) 701 - 717,   2006年07月01日, Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10β), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10β and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10β and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10β mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development. © 2006 The Authors Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
  • Cell-free expression systems for eukaryotic protein production
    Yaeta Endo, Tatsuya Sawasaki, Curr. Opin. Biotech., 17, (4) 373 - 380,   2006年08月01日, Following the success of genome sequencing projects, attention has now turned to studies of the structure and function of proteins. Although cell-based expression systems for protein production have been widely used, they have certain limitations in terms of the quality and quantity of the proteins produced and for high-throughput production. Many of these limitations can be circumvented by the use of cell-free translation systems. Among such systems, the wheat germ based system is of special interest for its eukaryotic nature; it has the significant advantage of producing eukaryotic multidomain proteins in a folded state. Several advances in the use of cell-free expression systems have been made in the past few years and successful applications of these systems to produce proteins for functional and structural biology studies have been reported. © 2006 Elsevier Ltd. All rights reserved.
  • In vitro selection of zinc finger DNA-binding proteins through ribosome display
    IHARA Hiroshi, MIE Masayasu, FUNABASHI Hisakage, TAKAHASHI Fumio, SAWASAKI Tatsuya, ENDO Yaeta, KOBATAKE Eiry, Biochem. and Biophys. Research Commun., 345, (3) 1149 - 1154,   2006年07月07日, DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique. © 2006 Elsevier Inc. All rights reserved.
  • A wheat germ cell-free system is a novel way to screen protein folding and function.
    Protein Science, 12, (6) 1216 - 1221,   2003年
  • Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate
    SAWASAKI Tatsuya, KAMURA Nami, KAMURA Nami, MATSUNAGA Satoko, SAEKI Mihoro, TSUCHIMOCHI Masateru, MORISHITA Ryo, ENDO Yaeta, ENDO Yaeta, ElsevierFEBS Letters, 582, (2) 221 - 228,   2008年01月23日, Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins. © 2007 Federation of European Biochemical Societies.
  • Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.
    TSUBOI Takafumi, TAKEO Satoru, IRIKO Hideyuki, JIN Ling, TSUCHIMOCHI Masateru, MATSUDA Shusaku, HAN Eun‐Taek, OTSUKI Hitoshi, KANEKO Osamu, SATTABONGKOT Jetsumon, UDOMSANGPETCH Rachanee, SAWASAKI Tatsuya, TORII Motomi, ENDO Yaeta, American Society For MicrobiologyInfection and immunity, 76, (4) 1702 - 1708,   2008年04月01日, One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 μg/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
  • A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis
    Hirotaka Takahashi, Hirotaka Takahashi, Akira Nozawa, Akira Nozawa, Akira Nozawa, Motoaki Seki, Kazuo Shinozaki, Kazuo Shinozaki, Kazuo Shinozaki, Kazuo Shinozaki, Yaeta Endo, Yaeta Endo, Yaeta Endo, Tatsuya Sawasaki, Tatsuya Sawasaki, Tatsuya Sawasaki, BioMed CentralBMC Plant Biology, 9,   2009年05月04日, Background. Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection. Results. Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity. Conclusion. In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.
  • Construction of a Protein Library of Arabidopsis Transcription Factors Using a Wheat Cell-Free Protein Production System and Its Application for DNA Binding Analysis
    Akira Nozawa, Akira Nozawa, Yuko Matsubara, Yoshinori Tanaka, Hirotaka Takahashi, Tatsuya Akagi, Motoaki Seki, Kazuo Shinozaki, Yaeta Endo, Yaeta Endo, Tatsuya Sawasaki, Tatsuya Sawasaki, Biosci. Biotechnol. Biochem., 73, (7) 1661 - 1664,   2009年08月19日, We created a protein library consisting of 647 Arabidopsis transcription factors (TFs) using a wheat cell-free system. The quality of proteins in the library was checked by binding assay of bZIP family proteins. Screening of TFs binding to 5′-regulatory regions of FLC and LFY was conducted using the library, and MYB67 and GBF1 were found to be binding factors.
  • Practical cell-free protein synthesis system using purified wheat embryos.
    Kazuyuki Takai, Tatsuya Sawasaki, Yaeta Endo, Nature Publishing Inc.Nature Protocols, 5, (2) 227 - 238,   2010年02月01日, Biochemical characterization of each gene product encoded in the genome is essential to understand how cells are regulated. The bottleneck has been and still is in how the gene products can be obtained. The wheat cell-free protein synthesis system we have developed is a powerful method for preparation of many different proteins at a time and also for preparation of large amounts of specific proteins for biochemical and structural analyses. Here, we show a method for preparation of the wheat embryo extract useful for the cell-free reactions, by which 5 ml of a high-activity extract is obtained in 4-5 d. We also describe the methods for small- and large-scale protein synthesis by hands-down operations with the use of mRNAs prepared by transcription of PCR products and pEU plasmids harboring the target cDNAs, which need 2-4 d excepting the time required for plasmid preparation. © 2010 Nature Publishing Group.
  • The wheat-germ cell-free expression system.
    Kazuyuki Takai, Tatsuya Sawasaki, Yaeta Endo, Current Pharmaceutical Biotechnology, 11, (3) 272 - 238,   2010年04月01日, We have made a dramatic improvement of the wheat cell-free protein synthesis system. The first key improvement is the method for preparation of the cell-free extract that is free of inhibitory factors of translation reaction. Additional improvements include a method for preparation of transcription-ready templates by PCR, an expression vector for the cell-free system, and the "bilayer" mode reaction method that is much more efficient than the batch mode method and at the same time easy to be performed by human hands and by liquid handling machines. We review here the history of the development and describe the protocols for the most handy "bilayer" method and a more efficient but complicated methods. Information on many examples and variations of the wheat cell-free protein synthesis methods already published elsewhere is then provided so that the readers can understand the power and potential applications of the methods. © 2010 Bentham Science Publishers Ltd.
  • Evaluating the Role of Rheumatoid Factors for the Development of Rheumatoid Arthritis in a Mouse Model with a Newly Established ELISA System.
    Yuki Tanaka, Yuki Tanaka, Hiroaki Komori, Shiro Mori, Yoshiko Soga, Takahito Tsubaki, Miho Terada, Tatsuhiko Miyazaki, Takahiro Fujino, Satoshi Nakamura, Hiroyuki Kanno, Tatsuya Sawasaki, Yaeta Endo, Masato Nose, Masato Nose, The Tohoku Journal of Experimental Medicine, 220, (3) 199 - 206,   2010年03月06日, Enzyme-linked immunosorbent assays (ELISA) have been widely used to determine quantitatively autoantibodies. However, the processes for the purification and immobilization of antigens in conventional ELISA methods include multiple steps, which have hampered the application for screening of autoantibodies. Here, we have developed a novel ELISA system using the plates pre-coated with glutathione casein to capture recombinant proteins fused to N-terminal glutathione S-transferase (GST). The GST-fused proteins were synthesized with the wheat germ cell-free protein production system. Thus, the present system combined the GST-capture ELISA with the cell-free protein production system, which allowed immobilization of the recombinant proteins with one-step purification. Using this ELISA method, we determined whether rheumatoid factors (RF), which have been considered as one of the representative disease - specific autoantibodies for rheumatoid arthritis (RA), were genetically associated with severity of arthritis in a mouse model for RA, MRL/Mp-lpr/lpr (MRL/lpr). GST-fused human IgG1-Fc (GST-Fc), synthesized with the robotic protein synthesizer, were used as reactants for RF. Serum samples for RF were prepared from 11 lines of a recombinant inbred mouse strain, MXH/lpr, which was established from intercrosses between MRL/lpr and non-arthritic C3H/HeJ-lpr/lpr (C3H/lpr) strains, composed of a different genomic recombination derived from the parental strains in each line. A correlation of RF titers with the severity of the arthritis in these lines was not significant, indicating genetic dissociation of RF from arthritis and that RF is not necessarily required for the development of RA. The present method may provide high-throughput screening for determining the disease-specific autoantibodies in autoimmune diseases. © 2010 Tohoku University Medical Press.
  • Paraquat toxicity induced by voltage-dependent anion channel 1 acting as an NADH-dependent oxidoreductase.
    SHIMADA Hiroki, HIRAI Kei‐ichi, SIMAMURA Eriko, HATTA Toshihisa, IWAKIRI Hiroki, MIZUKI Keiji, HATTA Taizo, SAWASAKI Tatsuya, SAWASAKI Tatsuya, MATSUNAGA Satoko, ENDO Yaeta, ENDO Yaeta, SHIMIZU Shigeomi, The Journal of Biological Chemistry, 284, (42) 28642 - 28649,   2009年10月16日, Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning areunreliable,andnumerousdeathshavebeenattributedinappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O2 .) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O2 . production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis
    Kazuhiro Matsuoka, Hiroaki Komori, Masato Nose, Masato Nose, Yaeta Endo, Yaeta Endo, Yaeta Endo, Yaeta Endo, Tatsuya Sawasaki, Tatsuya Sawasaki, Tatsuya Sawasaki, Tatsuya Sawasaki, American Chemical SocietyJournal of proteome research, 9, (8) 4264–4273 - 4273,   2010年08月06日, Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 μL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10000 in 25 μL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms. © 2010 American Chemical Society.
  • In vitro dissection revealed that the kinase domain of wheat RNA ligase is physically isolatable from the flanking domains as a non-overlapping domain enzyme
    MAKINO Shin−ichi, MAKINO Shin−ichi, SAWASAKI Tatsuya, SAWASAKI Tatsuya, SAWASAKI Tatsuya, ENDO Yaeta, ENDO Yaeta, ENDO Yaeta, TAKAI Kazuyuki, TAKAI Kazuyuki, TAKAI Kazuyuki, Elsevier Inc.Biochemical and Biophysical Research Communications, 397, (4) 762 - 766,   2010年07月01日, Wheat RNA ligase contains 5'-hydroxyl kinase, 2',3'-cyclic phosphate 3'-phosphodiesterase, and 5'-phosphate 2'-phosphate-3'-hydroxyl RNA ligase activities in a 110-kDa polypeptide. Taking advantage of a wheat cell-free protein production system, we prepared various fragments containing a part of the enzyme. The method allowed us to check the activities of the fragments rapidly, eliminating the time-consuming cloning and sequencing steps for the expression of the fragment proteins. The results showed that each of the three activities can be assigned to a non-overlapping domain that does not require the presence of the other part(s) of the enzyme for its activity. This contrasts to the case of yeast tRNA ligase, in which the central kinase domain has been suggested to require to be tethered to one of the flanking domains for its activity. © 2010 Elsevier Inc.
  • Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins
    Satoko Matsunaga, Kazuhiro Matsuoka, Kouhei Shimizu, Yaeta Endo, Yaeta Endo, Yaeta Endo, Yaeta Endo, Tatsuya Sawasaki, Tatsuya Sawasaki, Tatsuya Sawasaki, Tatsuya Sawasaki, BioMed Central Ltd.BMC Biotechnology, 10,   2010年06月04日, Background: Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.Results: The histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification (BISOP) method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase (CSK) obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots.Conclusion: We demonstrated that the newly developed BISOP method is very useful for obtaining high quality, tag-free recombinant proteins, produced using the cell-free system, for biochemical and structural analyses. © 2010 Matsunaga et al; licensee BioMed Central Ltd.
  • Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling
    Chidananda N. Kanchiswamy, Chidananda N. Kanchiswamy, Chidananda N. Kanchiswamy, Hirotaka Takahashi, Hirotaka Takahashi, Stefano Quadro, Massimo E. Maffei, Simone Bossi, Cinzia Bertea, Simon A. Zebelo, Atsushi Muroi, Atsushi Muroi, Nobuaki Ishihama, Hirofumi Yoshioka, Wilhelm Boland, Junji Takabayashi, Yaeta Endo, Tatsuya Sawasaki, Gen Ichiro Arimura, Gen Ichiro Arimura, BioMed Central Ltd.BMC Plant Biology, 10,   2010年05月26日, Background: Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.Results: To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.Conclusions: These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses. © 2010 Kanchiswamy et al; licensee BioMed Central Ltd.
  • Ca2+-dependent protein kinases and their substrate HsfB2a are differently involved in the heat response signaling pathway in Arabidopsis
    Chidananda Nagamangala Kanchiswamy, Chidananda Nagamangala Kanchiswamy, Chidananda Nagamangala Kanchiswamy, Atsushi Muroi, Atsushi Muroi, Massimo E. Maffei, Hirofumi Yoshioka, Tatsuya Sawasaki, Gen Ichiro Arimura, Gen Ichiro Arimura, Plant Biotechnology, 27, (5) 469 - 473,   2010年12月01日, Little is known about the mechanisms by which Ca2+-binding sensory proteins direct the plant heat shock (HS) response. Since two Ca2+-dependent protein kinases (CPK3 and CPK13) were recently shown to phosphorylate the heat shock transcription factor HsfB2a, we assessed in the current study whether these kinases are also involved in HS signal transduction, by monitoring the transcriptional profile of HS protein (Hsp) family genes in Arabidopsis Col-0 plants (WT) and the corresponding mutants. Both with and without HS, the gene transcript levels of Hsp70, Hsp101, Hsp17.4-CIII and Hsp15.7-CI were found to be lower in cpk3 and cpk13 mutants compared to WT, resulting in the impairment of basal thermotolerance in the mutants. To determine the in vivo function of CPKs, CPK3/13 and their substrate HsfB2a (heat shock transcription factor) were co-expressed as cofactors for the transient expression of a reporter (GUS) gene under the control of heat shock element (HSE) in Nicotiana benthamiana leaves. However, CPK3/13-phosphorylated HsfB2a did not function in the suppression/activation of HSE-promoted expression in the transient expression system. Implications for possible signal trafficking via CPKs and Hsfs are discussed. © 2010 The Japanese Society for Plant Cell and Molecular Biology.
  • Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules
    MAKINO Shin−ichi, MAKINO Shin−ichi, SAWASAKI Tatsuya, SAWASAKI Tatsuya, SAWASAKI Tatsuya, ENDO Yaeta, ENDO Yaeta, ENDO Yaeta, TAKAI Kazuyuki, TAKAI Kazuyuki, TAKAI Kazuyuki, Elsevier Inc.Biochemical and Biophysical Research Communications, 404, (4) 1050 - 1054,   2011年01月28日, Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5'-kinase, and 2',3'-cyclic phosphate 3'-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5'-tri/diphosphate RNAs is dependent on ATP, a 2'-phosphate-3'-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5' terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5'-phosphate and 2'-phosphate-3'-hydroxyl ends. Two RNA molecules having 5'-hydroxyl and 2',3'-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation. © 2010 Elsevier Inc.
  • Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system
    D. Tadokoro, S. Takahama, K. Shimizu, S. Hayashi, Y. Endo, Y. Endo, Y. Endo, T. Sawasaki, T. Sawasaki, T. Sawasaki, Mecmillan Publishers limitedCell Death and Disease, 1,   2010年10月01日, Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates. © 2010 Macmillan Publishers Limited All rights reserved.
  • Arabidopsis CPK3 plays extensive roles in various biological and environmental responses
    Gen Ichiro Arimura, Gen Ichiro Arimura, Tatsuya Sawasaki, Plant Signaling and Behavior, 5, (10) 1263 - 1265,   2010年10月01日, Plant Ca 2+-dependent protein kinase (CPK) signaling is involved in a wide array of intracellular signaling pathways involved in stomatal movement and plant adaptation to various environmental challenges including drought, salt and cold stress. Arabidopsis CPK3 appears to be extensively involved in such a wide range of aspects, and has been shown to function in mediating the signaling following Ca 2+ influx after insect herbivory. The results reveal the involvement of CPK3 in the herbivory-induced signaling network through phosphorylating the substrate target HsfB2a (heat shock transcription factor) for transcriptional activation of the plant defensin gene PDF1.2. Proteomic studies based on the cell-free protein production system allowed us to mine CPK3 targets more extensively and clarify the nature of multifunctional CPK3. © 2010 Landes Bioscience.
  • Wheat germ cell-free protein production system for post-genomic research
    Elsevier B.V.New Biotechnology, in press,   2010年

書籍等出版物

講演・口頭発表等

  • コムギ無細胞系を基盤としたタンパク質アレイによる、がん抑制タンパク質PTENのユビキチン化および分解に関わる新規なE3 ligaseの同定
    高橋 宏隆, 安岡 佐起, 竹田 浩之, 澤崎 達也, 第86回日本生化学会大会,   2013年09月13日, 日本生化学会
  • Establishment of robust assay platform based on cell-free protein array to analyze ubiquitin-mediated signal transductions
    The Ubiquitin System: From Basic Science to Drug Discovery (A2),   2014年01月08日, Keystone Sympodia
  • 創薬等プラットフォームにおけるコムギ無細胞基盤膜蛋白質生産と高親和膜蛋白質抗体作成技術
    澤崎 達也、竹田 浩之, 第13回日本蛋白質科学会年会,   2013年06月12日, 招待有り, 日本蛋白質科学会

作品

  • 小麦無細胞発現系の開発・改良および多検体の発現・可溶性検定
      2006年 - 2006年
  • 小麦胚芽抽出液の改良及び無細胞タンパク質合成の応用技術の開発
      2005年 - 2005年
  • 無細胞タンパク質合成系の保健衛生および畜産分野への応用
      2005年 - 2005年
  • 無細胞タンパク質合成法を用いる構造ゲノム科学基盤技術の確立
      2005年 - 2005年

特許

  • ハイスループット合成システム及び該システムを自動で行うための合成装置, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 森下 了, 佐伯 美帆呂, 佐藤 智久, 北本 綾, JP2004005912, WO2004-097014
  • ハイスループット合成システム及び該システムを自動で行うための合成装置, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 森下 了, 佐伯 美帆呂, 佐藤 智久, 北本 綾, 特願2005-505885, 特許第4643444号, 4643444
  • ポリメラーゼ阻害剤の阻害活性の測定方法, 森下 了, 澤崎 達也, 遠藤 弥重太, 梁 明秀, 山本 直樹, 特願2012-126648, 特開2015-156805
  • ビオチン化タンパク質の調製方法及び該タンパク質を用いた検出方法, 遠藤 弥重太, 澤崎 達也, 松原 祐子, 特願2006-182785, 特開2007-199047, US 7674593
  • 自己免疫疾患に関与するタンパク質の解析方法及び該疾患の検査方法, 澤崎 達也, 遠藤 弥重太, 石上 友章, 青木 一郎, JP2011076450, WO2012-067165
  • 関節リウマチに関与するタンパク質の解析方法及び該疾患の検査方法, 澤崎 達也, 遠藤 弥重太, 石上 友章, 青木 一郎, 中井 謙太, 特願2010-256401, 特開2012-107964
  • マラリアワクチン, 坪井 敬文, 鳥居 本美, 澤崎 達也, 遠藤 弥重太, 特願2010-164228, 特開2013-209291
  • 生理活性タンパク質に対する薬剤の新規ハイスループットスクリーニング法, 遠藤 弥重太, 澤崎 達也, JP2004013071, WO2005-024428, US7435538
  • 抗原検索法及び抗体検出法, 堤 寛, 水谷 泰嘉, 遠藤 弥重太, 澤崎 達也, 松岡 和弘, 特願2010-079277, 特開2011-209224
  • 最適な抗ウイルス剤の選択方法, 遠藤 弥重太, 澤崎 達也, 山本 直樹, 梁 明秀, 特願2008-154747, 特開2011-172486
  • 翻訳効率制御活性を有するヌクレオチド配列及びその利用, 遠藤 弥重太, 澤崎 達也, JP2002013756, WO2003-056009
  • 翻訳効率制御活性を有するヌクレオチド配列及びその利用, 遠藤 弥重太, 澤崎 達也, 特願2003-556526, 特許第3701292号, 3701292
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2005-355513, 特開2006-136330
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2005-355513, 特開2006-136330, 特許第4267621号, 3768190
  • タグとしての新規用途, 遠藤 弥重太, 澤崎 達也, 嘉村 奈美, 松原 祐子, JP2006312715, WO2007-000972
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 田中 理博, 森下 了, 佐伯 美帆呂, 特願2005-288613, 特開2007-097438
  • 無細胞タンパク質合成方法を用いた抗体検出方法及び特定タンパク質のスクリーニング方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 土持 政照, 松原 祐子, 特願2004-335392, 特開2008-035701
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 森下 了, 佐伯 美帆呂, 特願2004-329794, 特開2008-029203
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2004-329798, 特開2008-029204
  • 無細胞タンパク質合成用細胞抽出液及び該抽出液の調製方法, 遠藤 弥重太, 澤崎 達也, 石塚 芳子, 特願2004-308827, 特開2007-320853
  • 指標物質の新規スクリーニング方法, 遠藤 弥重太, 澤崎 達也, JP2004015122, WO2005-035780
  • 抗原物質の製造方法, 遠藤 弥重太, 坪井 敬文, 鳥居 本美, 澤崎 達也, JP2004013918, WO2005-030954
  • タンパク質チップ作製用試薬, 遠藤 弥重太, 澤崎 達也, 特願2006-519260, 特表2007-528718
  • 翻訳効率制御活性を有する核酸塩基配列及びその利用, 澤崎 達也, 遠藤 弥重太, 嘉村奈美, 特願2004-227866, 特開2006-042676
  • 無細胞タンパク質合成系で作られた蛍光を利用した効率的なタンパク質の機能解析スクリーニングの方法, 小林 民代, 遠藤 弥重太, 澤崎 達也, 特願2004-121886, 特開2005-308412
  • 自動蛋白質合成方法及びそれを行うための装置, 遠藤 弥重太, 澤崎 達也, JP2004001364, WO2004-070047
  • ハイスループット合成システム, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2003-281500, 特開2006-042601
  • 単鎖抗体およびその利用, 遠藤 弥重太, 川崎 平康, 澤崎 達也, JP2003009140, WO2004-009639
  • 単鎖抗体およびその利用, 遠藤 弥重太, 川崎 平康, 澤崎 達也, 特願2004-522758, 特許第4330532号
  • 無細胞タンパク質合成反応液、その調製方法及びそれを用いたタンパク質合成方法, 遠藤 弥重太, 川崎 平康, 澤崎 達也, JP2003002313, WO2003-072796
  • 無細胞タンパク質合成用転写鋳型の設計および構築、並びにこれを用いる希釈バッチ方式コムギ胚芽無細胞タンパク質合成法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, JP2001007357, WO2002-018586
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, JP2001007356, WO2002-024939
  • 無細胞タンパク質合成方法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2002-529531, 特許第3768190号
  • 無細胞タンパク質合成用転写鋳型の設計および構築、並びにこれを用いる希釈バッチ方式コムギ胚芽無細胞タンパク質合成法, 遠藤 弥重太, 澤崎 達也, 小笠原 富夫, 特願2002-522493, 特許第4762481号
  • タンパク質合成方法, 澤崎 達也, 小笠原 富夫, 遠藤 弥重太, 特願2001-238697, 特開2003-047496
  • 標識化単鎖抗体およびその利用, 4330532
  • 無細胞系合成システム, 10-0911516
  • 無細胞タンパク質合成反応液、その調整方法及びそれを用いたタンパク質合成方, US7273615

競争的資金

  • コムギ無細胞産生抗原を用いたサメナノボディ作成の基盤技術開発
    文部科学省, 科学研究費補助金(挑戦的萌芽研究), 澤崎 達也
  • 無細胞蛋白質アレイによる炎症カスパーゼ基質シグナル伝達分子の網羅的同定と解析
    文部科学省, 科学研究費補助金(基盤研究(B)), 澤崎 達也, I.カスパーゼ1により切断されるシグナル伝達タンパク質(sTMPおよびPK)の探索1) sTMPおよびPKプロテインアレイの構築申請者らが3年間費やして構築した約1000種類のシグナル伝達タンパク質cDNAクローン[300種類のプロテインキナーゼ(PK)および700種類の1回膜貫通領域タンパク質(sTMP)]を用いて、N末端にFlagタグ、C末端にビオチン化した組換えプロテインアレイ(NCタグプロテインアレイ)を合成し、超低温フリーザーに保存することができた。また、一部については、従来既に確認している様に、カスパーゼ3で切断されることを確認した。これにより、合成したプロテインアレイは基質として十分機能することがわかった。2)カスパーゼ1により切断されるシグナル伝達タンパク質の検出上記で作成したNCタグシグナル伝達プロテインアレイから、カスパーゼ1の基質として知られているインターフェロン1βを用いて、市販されているカスパーゼ1を用いて切断が検出できるかどうか調べた。その切断検出方法には、アルファスクリーン法を利用した。その結果、カスパーゼ1がインターフェロン1βの切断を示すシグナルの低下を検出することができた。この反応を従来のイムノブロット法でも確認し、カスパーゼ1の切断もアルファスクリーン法で検出できることがわかった。この事は、カスパーゼ1基質の網羅的な探索が可能な準備ができたことを意味する。
  • 無細胞蛋白質アレイによるポリユビキチン鎖依存シグナル伝達経路の網羅的同定と解析
    文部科学省, 科学研究費補助金(新学術領域研究(研究領域提案型)), 澤崎 達也, I.種々のポリユビキチン鎖への相互作用検出技術の確立予備実験的には成功していたポリユビキチン鎖に結合するタンパク質を高感度に検出できる技術の最適化を行った。その結合検出方法には、アルファスクリーン法を利用した。その原理は、384穴プレート中で、Flagタグでラベルされたユビキチン鎖(モノUb、ダイUb-Ub、テトラUb-Ub-Ubのいずれか)とビオチン化プロテインアレイタンパク質を反応させて、その後ドナービーズとアクセプタービーズを加えて反応させる。もし、ポリユビキチン鎖との相互作用反応が起これば、抗Flag抗体を介して双方のビーズが近接する。このような状態でドナービーズを励起すると、このエネルギーが近接したアクセプタービーズと反応し蛍光を発する。しかし、相互作用が起こらなければ、双方のビーズが近接できず、蛍光を発することができない。本手法を用いて、既知に報告されているA20タンパク質のZF7ドメインと直鎖状型ポリユビキチン鎖との結合を検出することができた。この事は、高感度にポリユビキチン鎖への相互作用反応が検出できる技術が確立できたことを意味する。II.アルファスクリーン法によるK63および直鎖状ポリユビキチン鎖と相互作用するシグナル伝達関連蛋白質の同定我々の研究室で進めきた手法を用いて、N末端にビオチン化した各種組換えプロテインアレイを構築することができた。最終的には、約400種のプロテインキナーゼ、250種のE3リガーゼおよび50種類の相互作用タンパク質からなるプロテインアレイを作成することに成功した。
  • 無細胞蛋白質アレイを基盤とした細胞がん化E3リガーゼの網羅的同定と解析
    文部科学省, 科学研究費補助金(新学術領域研究(研究領域提案型)), 澤崎 達也, I. ビオチン化RING-type E3リガーゼプロテインアレイの作製既に完成している152種類のRING-type E3リガーゼに加えて、申請者らが既に保有している1万5千種類のヒト完全長cDNAライブラリーを用いて、予測された約200種類のRING-type E3リガーゼ遺伝子のcDNAクローンを選択した。それらcDNAクローンから、スプリットPCR法により転写・翻訳用の鋳型を作製し、N末端にビオチン化した組換えプロテインアレイを合成し、現在、224種類のRING-type E3リガーゼプロテインアレイを構築することに成功し、超低温フリーザーに保存している。II. アルファスクリーン法によるがん化抑制因子および促進因子と結合するE3リガーゼ分子の同定上記で作成したビオチン化E3リガーゼプロテインアレイを、コムギ無細胞系で合成したFlagタグをN末端に付加した4種類のがん促進蛋白質(p53、LKB1、CYLD、PTEN)と結合するE3リガーゼのスクリーニングを行った。相互作用の検出方法には、アルファスクリーン法を利用した。その結果、それぞれのがん抑制蛋白質と相互作用するE3リガーゼを2~3種類同定することに成功した。また、その一部に関しては、in vitroでのE3リガーゼ依存的なユビキチン化が起こることを明らかとした。この事は、本スクリーニングシステムが基質特異的なE3リガーゼの探索・同定に有効であることを示している。
  • 蛋白アレイを用いた関節リウマチの新たな抗シトルリン化蛋白抗体の網羅的検索
    文部科学省, 科学研究費補助金(挑戦的萌芽研究), 大村 浩一郎, 抗環状シトルリン化ペプチド抗体(抗CCP抗体)は関節リウマチ(RA)の特異的自己抗体であるが、早期RAの約半数は抗CCP抗体陰性であり、診断がしばしば困難であるため抗CCP抗体陰性RAの特異的な診断マーカーが求められている。今回我々はAlphaScreen法と呼ばれる自己抗体の網羅的スクリーニング法を用いて抗CCP抗体陰性RA血清中にみられる未知のシトルリン化蛋白抗体のスクリーニングを行った。2000種類あまりのシトルリン化蛋白のうち、14個の未知のシトルリン化蛋白に対する反応がみられ、そのうち2個が頻度は少ないながら新規自己抗体であることが明らかになった。
  • 無細胞プロテインアレイを用いたカスパーゼ依存シグナル伝達経路の網羅的同定
    文部科学省, 科学研究費補助金(基盤研究(B)), 澤崎 達也, コムギ無細胞タンパク質合成システムにより構築した無細胞プロテインアレイを用いて、カスパーゼ3に切断されるプロテインキナーゼの網羅的なスクリーニングを行い、新規に30種類の基質プロテインキナーゼを見出した。切断部位の多くは、N末もしくはC末側のタンパク質の調節領域に位置していた。実際、いくつかの基質の個別解析により、カスパーゼ3の切断はタンパク質機能を調整していることが分かった。
  • 難治性内因性ぶどう膜炎における網羅的自己抗体解析研究
    文部科学省, 科学研究費補助金(基盤研究(C)), 大野 重昭, 本研究では我々が開発した自己抗体検出システムを用いて難治性内因性ぶどう膜炎の自己抗体を網羅的に検索・同定した。フォークト-小柳-原田病患者を3プール群に分けて検討した結果、患者3プール群すべてにおいて、健常コントロール群に比べて1.5倍以上の産生を示すタンパク質を10種類同定した。ベーチェット病においても同様に患者プール群すべてにおいて、健常コントロール群に比べて1.5倍以上の産生を示すタンパク質を6種類同定した。これら同定したタンパク質のうち、3種類は両疾患において共通であった。
  • カスパーゼをモデルとした蛋白質代謝ネットワーク解明に向けた基礎的技術開発
    文部科学省, 科学研究費補助金(萌芽研究), 澤崎 達也, 蛋白質は,細胞内もしくは細胞外でプロテアーゼにより“代謝"され,ペプチドホルモンなどの機能ペプチドとしてある種の生命現象の制御に関与していることが知られている。最近では、ホルモン機能以外にも,転写因子に直接結合することにより転写制御を行うペプチドの発見など,従来では予想されていなかった蛋白質の“代謝"産物ペプチドが多種多様な機能を有する可能性が示唆されている。これを裏付けるかのように,高等動物や植物のゲノム上には,プロテアーゼは500種類以上からなる大きなファミリーを形成しており,この数はシグナル伝達の中心的な役割を示すプロテインカイネースや転写因子ファミリーに匹敵する。この事は,高等生物ゲノムがコードする数万種類の蛋白質は,多種のプロテアーゼが関与した機構により代謝され,その産物(ペプチド)が生命現象制御に広く関与した,“蛋白質代謝ネットワーク"を形成している可能性を示唆するものである。本申請はこのようなネットワークを網羅的に調べることができる技術や方法論の開発を目指すものである。この技術の利用は,“代謝"される蛋白質情報だけでなく,機能を有したペプチドの同定が可能となり,まだリガンドが未同定なオーファン受容体との相互作用や,代謝ペプチドラィブラリーの構築への利用などの応用研究,『蛋白質の分解とその運命』という古くて新しい領域の解明につながるものと期待される。しかし,これらの方法論の構築には,ハイスループットな基質蛋白質合成と切断活性の検出法の構築が必要である。本年度は、昨年度見出した新規カスパーゼ3切断プロテインカイネースの切断部位および、アポトーシス誘導時の細胞内での切断を確認した.
  • ベーチェット病のインフリキシマブ治療不応答性因子の解明
    文部科学省, 科学研究費補助金(挑戦的萌芽研究), 水木 信久, 本研究では、日本人ベーチェット病患者を対象に、インフリキシマブ不応答性に関わる因子の同定を試みた。網羅的な自己抗体解析の結果、インフリキシマブ不応答性に関与を示唆するタンパク質が複数検出された。また、ゲノムワイド関連解析により得られた遺伝情報をもとに遺伝因子の検索を行った結果、インフリキシマブ不応答性への関与を示唆する複数の遺伝子が検出された。
  • 膜型増殖因子のER/核膜オートトランスロケーション分子機構解明とがん特性診断応用
    文部科学省, 科学研究費補助金(挑戦的萌芽研究), 東山 繁樹, 膜型細胞増殖因子proHB-EGFやproAREGは,正常細胞では細胞膜に局在するが、悪性度の高いがん細胞には、直接ER/核膜に移行する(これをオートトランスロケーションと呼ぶ)例があり、有意な抗がん剤抵抗性を示す。本研究では、proAREGのER/核膜オートトランスロケーションと抗がん剤抵抗性獲得をin vivo解析により、実証した。また、その分子機構の一端として、C-末端ペプチドの切断を提唱し、責任プロテアーゼの探索を行った。本研究は、抗がん剤感受性診断法開発に向けて、有用性とその意義を示した。
  • マラリア原虫メロゾイト先端部小器官分子に対するヒト赤血球レセプターの網羅的同定
    文部科学省, 科学研究費補助金(基盤研究(A)), 坪井 敬文, マラリア原虫メロゾイトの先端部小器官に局在する新規分子を同定するため、メロゾイト期に発現が予想される分子193種類をコムギ無細胞系にて合成し、すべてに対する抗体を作製した。その抗体の内、メロゾイト先端部小器官と反応するものを間接蛍光抗体法にて探索し、29種類の新規メロゾイト先端部小器官分子を同定した。それらの内、マイクロネームに局在するGAMAの赤血球レセプターの性状を明らかにした。さらに、約500種類の赤血球分子を網羅するタンパク質アレイを作製し、赤血球に結合するロプトリータンパク質のレセプター分子を同定した。
  • 植物の環境適応・被食防衛に応答するカルシウム依存リン酸化・ユビキチン化制御機構
    文部科学省, 科学研究費補助金(若手研究(B)), 有村 源一郎, 食害や環境ストレスによって活性されるカルシウム依存性タンパク質リン酸化酵素(Calcium-Dependent Protein Kinase:CPK)とそれらの基質タンパク質に着目し、食害および高温ストレスに曝されたシロイヌナズナにおける遺伝子転写制御の分子機構の解明を試みた。CPK変異体を用いた解析等により、CPK3とCPK13による基質タンパク質(HsfB2a[転写因子]など)のリン酸化は防御遺伝子と熱ショック遺伝子の発現誘導において重要であり、これらのCPKはリン酸化する基質ターゲットを変化させ、様々なストレス環境下において多様な役割を担うことが示唆された。
  • 無細胞翻訳系によるセレノメチオニン標識タンパク質の生産とX線結晶構造解析への応用
    文部科学省, 科学研究費補助金(基盤研究(C)), 堀 弘幸, 無細胞翻訳系によるセレノメチオニン(SeMet)標識を、クラゲ蛍光タンパク質(GFP)および菌ジヒドロ葉酸還元酵素(DHFR)をモデルタンパク質として検討した。その結果、いずれのタク質も、メチオニン(Met)を用いた通常の合成量に匹敵する量で合成が可能であった。合成したパク質中のSeMet置換は、液体クロマトグラフィ-質量分析により確認した。全液体クロマトグを精査しても、内在性Metによる該当ペプチドを検出できなかったことから、SeMet置換量は100%に近いことが確認された。また、この過程でSeMet標識タンパク質を、-80℃で6ヶ月間してもSeMet基が酸化を受けないことも確認された。GFPの発色団形成は、通常のメチオニン標識よりもややスローであったが、その蛍光スペクはSeMet-GFPとMet-GFP間でよい一致をしめした。この結果は、少なくとも発色団近傍のタク質立体構造形成にSeMet標識が何も影響を与えないことを示している。また、DHFRの場合、残基の位置が触媒機構上重要なMet20-ループに含まれることから、触媒活性への影響が懸念さが、これもほぼ同等の活性を有することが確認された。これら、モデルタンパク質による標識の検討を終え、X線構造解析に着手しているtRNA修飾のSeMet導入部位検索に利用した。現在、本酵素は1.5Å解像度で構造精密化を行なっている。よって、無細胞翻訳系により、高効率でSeMet標識が可能であることが確認され、x線結晶構造解析への応用が可能なものと考えている。本研究の一部は、日本生化学会および分子生物学会において発表し、解析中の遺伝子のクローニングおよびドメイン構造、活性に必須なアミノ酸残基についてはすでにいくつかの論文として公表した。構造解析の詳細については、平成15年度以降、順次、論文として発表する予定である。
  • 植物細胞死機構に関する研究
  • 無細胞系を用いた網羅的タンパク質機能解析
    新技術・新分野創出のための基礎研究推進事業

教育活動情報

担当経験のある科目

  • タンパク質科学, 愛媛大学大学院理工学研究科
  • 応用化学実験, 愛媛大学工学部
  • 分子生物学, 愛媛大学工学部


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.