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研究生: 鄭皓蔚
Hao-Wei Cheng
論文名稱: 以色胺酸及FlAsH組成之凋亡蛋白酶3螢光感測多肽
FlAsH Labeled Peptide with Tryptophan Residues as a Caspase-3 Sensor
指導教授: 李賢明
Hsein-Ming Lee
謝發坤
Fa-Kuen Shieh
口試委員:
學位類別: 碩士
Master
系所名稱: 理學院 - 化學學系
Department of Chemistry
論文出版年: 2015
畢業學年度: 103
語文別: 中文
論文頁數: 116
中文關鍵詞: 感測器凋亡蛋白酶3螢光
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  • 本論文是以色胺酸對螢光團螢光淬滅的特性為基礎,設計並合成蛋白酶活性
    感測器 FlAsH-3W2F-TCǶ利用一系列 FlAsH 螢光染料標示胜肽的螢光性質進行研究,發現帶有芳香環側鏈的胺基酸中,相較於阻胺酸與苯Ҙ胺酸,色胺酸對FlAsH 螢光染料的螢光有最好的淬滅效果;胜肽上的色胺酸數量越多對螢光的淬滅效果也越好Ƕ色胺酸對於螢光染料的淬滅是根據光誘發電子轉移,透過螢光分子與色胺酸之間碰撞所形成暫時的分子軌域,提供電子轉移的通道,使螢光團激發的能量得到鬆弛Ƕ在以 Trypsin 為模型的蛋白酶活性實驗中,感測器水解前後有將近 9 倍的螢光強度增加;在 Caspase-3 目標蛋白酶活性實驗中,也觀測到明顯的螢光增強Ƕ以色胺酸做為淬滅團在螢光感測器上的應用是可行的。


    We synthesized a Caspase-3 sensor, FlAsH-3W2F-TC, whose fluorescence was quenched by tryptophan inside the peptidyl sensor backbone. By studying a series of FlAsH-labeled peptide sequences, tryptophan containing peptide sequence showed higher quenching ability than histidine and phenylalanine peptide sequences; the more tryptophan the peptide sequence contains, the higher quenching efficiency we get. The quenching phenomena is based on photo-induced electron transfer(PET). The excited fluorophore relax by conducting an electron through temporary collisional molecular orbital. In trypsin model experiment, our sensor showed ~9-fold fluorescence increasing after hydrolysis; in Caspase-3 experiment, our sensor also showed significant fluorescence increasing. Finally, the application of tryptophan as a natural quencher in peptidyl fluorescent sensor is feasible and potential.

    目錄 中文摘要 ---------------------------------------------------------------------------- I 英文摘要 ---------------------------------------------------------------------------- II 致謝 ---------------------------------------------------------------------------- III 目錄 ---------------------------------------------------------------------------- IV 圖目錄 ---------------------------------------------------------------------------- VIII 表目錄 ---------------------------------------------------------------------------- XV 第一章 緒論 ---------------------------------------------------------------------- 1 1-1 細胞凋亡(Apoptosis) ------------------------------------------------- 1 1-1-1 外在路徑誘發細胞凋亡(Extrinsic pathway of cell apoptosis) - 2 1-1-2 內在路徑誘發細胞凋亡(Intrinsic pathway of cell apoptosis) -- 3 1-1-3 藥物引發細胞凋亡 ---------------------------------------------------- 3 1-1-4 亡蛋白酶(Caspase) --------------------------------------------------- 4 1-1-4-1 細胞凋亡蛋白酶-3 (Caspase-3) ------------------------------------ 6 1-2 螢光生物感測 ---------------------------------------------------------- 6 1-2-1 生物感測 ---------------------------------------------------------------- 6 1-2-1-1 概述 ---------------------------------------------------------------------- 6 1-2-1-2 辨識元件(Recognition unit) ----------------------------------------- 6 1-2-1-3 訊號轉換器(Signal transducer) ------------------------------------- 7 1-2-2 螢光發光團(Fluorophore) ------------------------------------------ 8 1-2-2-1 原理 ---------------------------------------------------------------------- 8 1-2-2-2 螢光染料 ---------------------------------------------------------------- 8 1-2-2-3 螢光蛋白質 ------------------------------------------------------------- 8 1-2-3 淬滅團(Quencher) ---------------------------------------------------- 9 V 1-2-3-1 原理 ---------------------------------------------------------------------- 9 1-2-3-2 Fröster 共振能量轉移(Fröster Resonance Energy Transfer, FRET) ------------------------------------------------------------------- 10 1-2-3-3 光誘發電子轉移 (Photoinduced Electron Transfer, PET) ----- 10 1-2-4 化學合成螢光生物感測 ---------------------------------------------- 11 1-2-5 基因表達螢光生物感測 ---------------------------------------------- 11 1-2-6 半基因表達合成螢光生物感測 ------------------------------------- 12 第二章 文獻回顧與設計構想 ------------------------------------------------- 13 2-1 凋亡蛋白酶的檢測方式 ---------------------------------------------- 13 2-1-1 比色法定量Caspase-3 活性 ------------------------------------------ 13 2-1-2 單一螢光團定量Caspase-3活性 ------------------------------------ 14 2-1-3 FRET 定量 Caspase-3 活性感測 ------------------------------------ 17 2-1-4 奈米粒子Caspase-3活性感測 --------------------------------------- 18 2-1-5 螢光蛋白質對Caspase-3活性感測 --------------------------------- 23 2-1-6 自聚集誘發螢光 (Aggregation-induced emission,AIE) 染料 Caspase-3 活性偵測器 ------------------------------------------------ 27 2-2 色胺酸對螢光團淬滅 ------------------------------------------------- 30 2-3 針對 Caspase-3 活性感測器的構想 ------------------------------- 36 2-4 實驗設計與現存感測器的差別比較 ------------------------------- 38 第三章 結果與討論 ------------------------------------------------------------- 39 3-1 胜肽合成 ---------------------------------------------------------------- 39 3-2 螢光染料標識胜肽 ---------------------------------------------------- 40 3-3 胜肽序列對 FlAsH 螢光淬滅效果比較 -------------------------- 41 VI 3-4 Trypsin 實驗 FlAsH 標識胜肽螢光恢復能力 ------------------- 43 3-5 Caspase-3 實驗螢光恢復能力 -------------------------------------- 45 3-6 Caspase-3 定性及定量 ------------------------------------------------ 47 3-6-1 SDS-page 定性 --------------------------------------------------------- 48 3-6-2 Caspase-3 活性測量 --------------------------------------------------- 49 3-6-2-1 pNA 校正曲線 --------------------------------------------------------- 49 3-6-2-2 Caspase-3 活性測量與計算 ------------------------------------------ 49 3-7 環境溫度對 Caspase-3 活性的迎想 -------------------------------- 50 3-8 溶液中蛋白質對 FlAsH-TC 螢光強度隨時間下降的影響 ----- 51 3-9 BSA 對 FlAsH-3W2F-TC 螢光的影響 ----------------------------- 52 第四章 結論 ---------------------------------------------------------------------- 54 第五章 實驗方法 ---------------------------------------------------------------- 55 5-1 實驗儀器 ---------------------------------------------------------------- 55 5-2 實驗藥品 ---------------------------------------------------------------- 55 5-3 實驗步驟 ---------------------------------------------------------------- 57 5-3-1 Caspase-3 感測器 ----------------------------------------------------- 57 5-3-1-1 固態胜肽合成 ---------------------------------------------------------- 57 5-3-1-2 胜肽純化 ---------------------------------------------------------------- 58 5-3-1-3 FlAsH 螢光標識 ------------------------------------------------------ 59 5-3-1-4 FlAsH 標釋胜肽定量 ------------------------------------------------ 60 5-3-1-5 蛋白質對感測器螢光增強實驗 ------------------------------------- 60 5-3-2 Caspase-3 表現與純化 ----------------------------------------------- 61 5-3-2-1 培養基配置 ------------------------------------------------------------- 61 5-3-2-2 pET23b-Casp3-His 質體放大及純化 ------------------------------- 61 5-3-2-3 Caspase-3 表現 -------------------------------------------------------- 62 5-3-2-4 凋亡蛋白酶活性測量 ------------------------------------------------- 63 5-3-3 溶液配置 ---------------------------------------------------------------- 64 參考文獻 ---------------------------------------------------------------------------- 65 附錄一 光譜資料 ---------------------------------------------------------------- 70 附錄二 Fluorecein / TAMRA FRET 對的感測器設計 --------------------- 80

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    參考文獻:
    (1) Elmore, S. Toxicologic Pathology. 2007, 35, 495.
    (2) Peterson, Q. P.; Goode, D. R.; West, D. C.; Botham, R. C.; Hergenrother, P.
    J. Nature Protocols. 2010, 5, 294.
    (3) Majno, G.; Joris, I. American Journal of Pathology. 1995, 146, 3.
    (4) Janicke, R. U.; Sprengart, M. L.; Wati, M. R.; Porter, A. G. Journal of
    Biological Chemistry 1998, 273, 9357.
    (5) Cohen, G. M. Biochemical Journal. 1997, 326, 1.
    (6) Fadok, V. A.; Savill, J. S.; Haslett, C.; Bratton, D. L.; Doherty, D. E.;
    Campbell, P. A.; Henson, P. M. Journal of Immunology 1992, 149, 4029.
    (7) Smith, C. A.; Farrah, T.; Goodwin, R. G. Cell 1994, 76, 959.
    (8) Ashkenazi, A.; Dixit, V. M. Science 1998, 281, 1305.
    (9) Green, D. R.; Reed, J. C. Science 1998, 281, 1309.
    (10) Fulda, S.; Debatin, K. M. Oncogene 2006, 25, 4798.
    (11) Liu, X. S.; Kim, C. N.; Yang, J.; Jemmerson, R.; Wang, X. D. Cell 1996, 86,
    147.
    (12) Zou, H.; Henzel, W. J.; Liu, X. S.; Lutschg, A.; Wang, X. D. Cell 1997, 90,
    405.
    (13) Yang, J.; Liu, X. S.; Bhalla, K.; Kim, C. N.; Ibrado, A. M.; Cai, J. Y.; Peng,
    T. I.; Jones, D. P.; Wang, X. D. Science 1997, 275, 1129.
    (14) Aas, T.; Borresen, A. L.; Geisler, S.; SmithSorensen, B.; Johnsen, H.;
    Varhaug, J. E.; Akslen, L. A.; Lonning, P. E. Nature Medicine 1996, 2, 811.
    (15) Wang, S. W.; Konorev, E. A.; Kotamraju, S.; Joseph, J.; Kalivendi, S.;
    Kalyanaraman, B. Journal of Biological Chemistry 2004, 279, 25535.
    (16) Nicolier, M.; Decrion-Barthod, A.-Z.; Launay, S.; Pretet, J.-L.; Mougin, C.
    Biology of the Cell 2009, 101, 455. 66

    (17) Belmokhtar, C. A.; Hillion, J.; Segal-Bendirdjian, E. Oncogene 2001, 20,
    3354.
    (18) Chae, H.-J.; Kang, J.-S.; Byun, J.-O.; Han, K.-S.; Kim, D.-U.; Oh, S.-M.;
    Kim, H.-M.; Chae, S.-W.; Kim, H.-R. Pharmacological Research 2000, 42, 373.
    (19) Pop, C.; Salvesen, G. S. Journal of Biological Chemistry 2009, 284, 21777.
    (20) Johansson, M. K.; Cook, R. M. Chemistry-a European Journal. 2003, 9,
    3466.
    (21) Ward, M. D. Chemical Society Reviews. 1997, 26, 365.
    (22) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata, Y.;
    Kanagawa, T.; Kurane, R. Analytical sciences : the international journal of the Japan
    Society for Analytical Chemistry 2001, 17, 155.
    (23) de Silva, A. P.; Moody, T. S.; Wright, G. D. Analyst 2009, 134, 2385.
    (24) Doose, S.; Neuweiler, H.; Sauer, M. ChemPhysChem 2005, 6, 2277.
    (25) Doose, S.; Neuweiler, H.; Sauer, M. ChemPhysChem 2009, 10, 1389.
    (26) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nature reviews.
    Molecular cell biology 2002, 3, 906.
    (27) Hoffmann, C.; Gaietta, G.; Bunemann, M.; Adams, S. R.; Oberdorff-Maass,
    S.; Behr, B.; Vilardaga, J. P.; Tsien, R. Y.; Eisman, M. H.; Lohse, M. J. Nature
    Methods. 2005, 2, 171.
    (28) Thornberry, N. A.; Bull, H. G.; Calaycay, J. R.; Chapman, K. T.; Howard, A.
    D.; Kostura, M. J.; Miller, D. K.; Molineaux, S. M.; Weidner, J. R.; Aunins, J.;
    Elliston, K. O.; Ayala, J. M.; Casano, F. J.; Chin, J.; Ding, G. J. F.; Egger, L. A.;
    Gaffney, E. P.; Limjuco, G.; Palyha, O. C.; Raju, S. M.; Rolando, A. M.; Salley, J. P.;
    Yamin, T. T.; Lee, T. D.; Shively, J. E.; Maccross, M.; Mumford, R. A.; Schmidt, J. A.;
    Tocci, M. J. Nature 1992, 356, 768.
    (29) Rotonda, J.; Nicholson, D. W.; Fazil, K. M.; Gallant, M.; Gareau, Y.; 67

    Labelle, M.; Peterson, E. P.; Rasper, D. M.; Ruel, R.; Vaillancourt, J. P.; Thornberry, N.
    A.; Becker, J. W. Nature structural biology 1996, 3, 619.
    (30) Han, Z. Y.; Hendrickson, E. A.; Bremner, T. A.; Wyche, J. H. Journal of
    Biological Chemistry 1997, 272, 13432.
    (31) Stennicke, H. R.; Renatus, M.; Meldal, M.; Salvesen, G. S. The Biochemical
    journal 2000, 350 Pt 2, 563.
    (32) Tewari, M.; Quan, L. T.; Orourke, K.; Desnoyers, S.; Zeng, Z.; Beidler, D.
    R.; Poirier, G. G.; Salvesen, G. S.; Dixit, V. M. Cell 1995, 81, 801.
    (33) Roy, A. C.; Saha, N.; Tan, S. M.; Kamarul, F. Z.; Ratnam, S. S.
    Electrophoresis 1992, 13, 396.
    (34) Gurtu, V.; Kain, S. R.; Zhang, G. Analytical Biochemistry. 1997, 251, 98.
    (35) Liu, J.; Bhalgat, M.; Zhang, C.; Diwu, Z.; Hoyland, B.; Klaubert, D. H.
    Bioorganic & Medicinal Chemistry Letters. 1999, 9, 3231.
    (36) Wang, Z.-Q.; Liao, J.; Diwu, Z. Bioorganic & Medicinal Chemistry Letters
    2005, 15, 2335.
    (37) Cai, S. X.; Zhang, H.-Z.; Guastella, J.; Drewe, J.; Yang, W.; Weber, E.
    Bioorganic & Medicinal Chemistry Letters 2001, 11, 39.
    (38) Bullok, K.; Piwnica-Worms, D. Journal of Medicinal Chemistry. 2005, 48,
    5404.
    (39) Bullok, K. E.; Maxwell, D.; Kesarwala, A. H.; Gammon, S.; Prior, J. L.;
    Snow, M.; Stanley, S.; Piwnica-Worms, D. Biochemistry 2007, 46, 4055.
    (40) Maxwell, D.; Chang, Q.; Zhang, X.; Barnett, E. M.; Piwnica-Worms, D.
    Bioconjugate Chem. 2009, 20, 702.
    (41) Boeneman, K.; Mei, B. C.; Dennis, A. M.; Bao, G.; Deschamps, J. R.;
    Mattoussi, H.; Medintz, I. L. Journal of the American Chemical Society 2009, 131,
    3828. 68

    (42) Prasuhn, D. E.; Feltz, A.; Blanco-Canosa, J. B.; Susumu, K.; Stewart, M. H.;
    Mei, B. C.; Yakovlev, A. V.; Loukov, C.; Mallet, J.-M.; Oheim, M.; Dawson, P. E.;
    Medintz, I. L. Acs Nano 2010, 4, 5487.
    (43) Kihara, T.; Nakamura, C.; Suzuki, M.; Han, S. W.; Fukazawa, K.; Ishihara,
    K.; Miyake, J. Biosensors & Bioelectronics. 2009, 25, 22.
    (44) Shi, Y.; Yi, C.; Zhang, Z.; Zhang, H.; Li, M.; Yang, M.; Jiang, Q. ACS
    applied materials & interfaces 2013, 5, 6494.
    (45) Xu, X.; Gerard, A. L. V.; Huang, B. C. B.; Anderson, D. C.; Payan, D. G.;
    Luo, Y. Nucleic Acids Research. 1998, 26, 2034.
    (46) Nagai, T.; Miyawaki, A. Biochemical and Biophysical Research
    Communications 2004, 319, 72.
    (47) Elphick, L. M.; Meinander, A.; Mikhailov, A.; Richard, M.; Toms, N. J.;
    Eriksson, J. E.; Kass, G. E. N. Analytical Biochemistry. 2006, 349, 148.
    (48) Sakamoto, S.; Terauchi, M.; Hugo, A.; Kim, T.; Araki, Y.; Wada, T.
    Chemical Communications. 2013, 49, 10323.
    (49) Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Chemical Communications.. 2009,
    4332.
    (50) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. Journal of
    the American Chemical Society 2012, 134, 17972.
    (51) Shi, H. B.; Zhao, N.; Ding, D.; Liang, J.; Tang, B. Z.; Liu, B. Organic &
    Biomolecular Chemistry 2013, 11, 7289.
    (52) Ding, D.; Liang, J.; Shi, H. B.; Kwok, R. T. K.; Gao, M.; Feng, G. X.; Yuan,
    Y. Y.; Tang, B. Z.; Liu, B. Journal of Materials Chemistry B 2014, 2, 231.
    (53) Marme, N.; Knemeyer, J. P.; Sauer, M.; Wolfrum, J. Bioconjugate
    Chemistry. 2003, 14, 1133.
    (54) Marme, N.; Knemeyer, J. P.; Wolfrum, J.; Sauer, M. Angewandte Chemie-International Edition. 2004, 43, 3798.
    (55) Togashi, D. M.; Szczupak, B.; Ryder, A. G.; Calvet, A.; O'Loughlin, M.
    Journal of Physical Chemistry A. 2009, 113, 2757.
    (56) Albert Griffin, B.; Adams, S. R.; Jones, J.; Tsien, R. Y. In Methods in
    Enzymology; Jeremy Thorner, S. D. E., John, N. A., Eds.; Academic Press: 2000; Vol.
    Volume 327, p 565.

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