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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊?伸 | |
| dc.contributor.author | Ching-Che Huang | en |
| dc.contributor.author | 黃敬哲 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:17:45Z | - |
| dc.date.available | 2014-07-28 | |
| dc.date.copyright | 2009-07-28 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-27 | |
| dc.identifier.citation | 參考資料
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Angewandte Chemie International Edition 37, 1518-1519 (1998). 22. Hampp, N. Bacteriorhodopsin as a Photochromic Retinal Protein for Optical Memories. Chemical Reviews 100, 1755 (2000). 23. Mathies, R., Lin, S., Ames, J. & Pollard, W. From femtoseconds to biology: mechanism of bacteriorhodopsin's light-driven proton pump. Annual Review of Biophysics and Biophysical Chemistry 20, 491-518 (1991). 24. Ebrey, T. Light energy transduction in bacteriorhodopsin. Thermodynamics of Membrane Receptors and Channels. MB Jackson, editor. CRC Press, Boca Raton, FL, 353–387 (1993). 25. Birge, R.R. et al. Revised assignment of energy storage in the primary photochemical event in bacteriorhodopsin. Journal of the American Chemical Society 113, 4327 (2002). 26. Brown, L., Bonet, L., Needleman, R. & Lanyi, J. Estimated acid dissociation constants of the Schiff base, Asp-85, and Arg-82 during the bacteriorhodopsin photocycle. Biophysical Journal 65, 124-130 (1993). 27. Lanyi, J. & Schobert, B. Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2′ intermediates of the photocycle. Journal of Molecular Biology 328, 439-450 (2003). 28. Rammelsberg, R., Huhn, G., Lubben, M. & Gerwert, K. Bacteriorhodopsin's Intramolecular Proton-Release Pathway Consists of a Hydrogen-Bonded Network? Biochemistry 37, 5001-5009 (1998). 29. Spassov, V., Luecke, H., Gerwert, K. & Bashford, D. pKa calculations suggest storage of an excess proton in a hydrogen-bonded water network in bacteriorhodopsin. Journal of Molecular Biology 312, 203-219 (2001). 30. Varo, G. & Lanyi, J. Kinetic and spectroscopic evidence for an irreversible step between deprotonation and reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Biochemistry 30, 5008-5015 (1991). 31. Zimanyi, L., Chang, M., Ni, B., Needleman, R. & Lanyi, J. The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements. Photochemistry and photobiology 56, 1049-1055 (1992). 32. Liu, S., Kono, M. & Ebrey, T. Effect of pH buffer molecules on the light-induced currents from oriented purple membrane. Biophysical Journal 60, 204-216 (1991). 33. Zimanyi, L. et al. Pathways of proton release in the bacteriorhodopsin photocycle. Biochemistry 31, 8535-8543 (1992). 34. Schobert, B., Brown, L. & Lanyi, J. Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base. Journal of Molecular Biology 330, 553-570 (2003). 35. Rouhani, S. et al. Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate. Journal of Molecular Biology 313, 615-628 (2001). 36. Dunn, R. et al. Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli. Journal of Biological Chemistry 262, 9246-9254 (1987). 37. Hohenfeld, I., Wegener, A. & Engelhard, M. Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli. FEBS letters 442, 198-202 (1999). 38. Kalmbach, R. et al. Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. Journal of Molecular Biology 371, 639-648 (2007). 39. Krebs, M., Mollaaghababa, R. & Khorana, H. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants. Proceedings of the National Academy of Sciences 90, 1987-1991 (1993). 40. Wang, W., Sineshchekov, O., Spudich, E. & Spudich, J. Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin*. Journal of Biological Chemistry 278, 33985-33991 (2003). 41. Shen, Y., Safinya, C., Liang, K., Ruppert, A. & Rothschild, K. Stabilization of the membrane protein bacteriorhodopsin to 140 C in two-dimensional films. Nature 366, 48-50 (1993). 42. Taneva, S., Caaveiro, J., Petkanchin, I. & Goni, F. Electrokinetic charge of the anesthetic-induced bR480 and bR380 spectral forms of bacteriorhodopsin. BBA-Biomembranes 1236, 331-337 (1995). 43. Bamberg, E., Dencher, N., Fahr, A. & Heyn, M. Transmembraneous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers. Proceedings of the National Academy of Sciences 78, 7502-7506 (1981). 44. Eisenbach, M. & Caplan, S. Interaction of purple membrane with solvents. II. Mode of interaction. Biochimica et biophysica acta 554, 281 (1979). 45. Hampp, N., Brauchle, C. & Oesterhelt, D. Bacteriorhodopsin wildtype and variant aspartate-96→ aspargine as reversible holographic media. Biophysical Journal 58, 83-93 (1990). 46. Birge, R. Photophysics and molecular electronic applications of the rhodopsins. Annual Review of Physical Chemistry 41, 683-733 (1990). 47. Yao, B. et al. Polarization holographic high-density optical data storage in bacteriorhodopsin film. Applied optics 44, 7344-7348 (2005). 48. Hampp, N., Brauchle, C. & Oesterhelt, D. Mutated bacteriorhodopsins: Competitive materials for optical information processing. MRS Bulletin 17 (1992). 49. Hampp, N. & Silber, A. Functional dyes from nature: Potentials for technical applications. Pure and applied chemistry 68, 1361-1366 (1996). 50. Miyasaka, T. & Koyama, K. Image sensing and processing by a bacteriorhodopsin-based artificial photoreceptor. Applied optics 32, 6371-6379 (1993). 51. Cherezov, V. & Caffrey, M. Membrane protein crystallization in lipidic mesophases. A mechanism study using X-ray microdiffraction. Faraday Discussions 136, 195-212 (2007). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42614 | - |
| dc.description.abstract | 中文摘要
細菌視紫紅質被發現在嗜鹽古細菌的紫膜 (purple membrane) 上，當嗜鹽古細菌遭遇到低氧氣以及高光照的環境，這樣的蛋白質就會被表現。其有七個穿膜 α 螺旋，並在蛋白質的中心以schiff base 形式連結 retinal 。接受光照時，蛋白質內部的發色基團 retinal 會產生形變，將氫離子從蛋白質的一端運輸至另一端，而在細胞膜的兩側形成一個氫離子的梯度。這樣的化學能梯度可藉由 ATP synthase 而達到 ADP 的磷酸化，以讓嗜鹽古細菌能在氧氣量較低的環境產生能量。細菌視紫紅質由於內部有一個發色基團，因此擁有特殊吸收光譜，光週期；又因為其在原物種內的高產量及穩定性，讓他成為一個很有潛力的生物感光材料。本實驗藉由電腦的序列分析，以及光譜測定，光週期測定和氫離子幫浦能力來測試 HmBRI ( Haloarcula marismortui BRI ) 的性質，確認其為細菌視紫質；並且發現另外幾個具有點突變之 HmBRI，其中 D94N 這個突變可以讓 HmBRI 在 E.coli 系統內有極高的表現量，且其對於熱和光穩定，也可處於無水環境下仍保有其特性。在定性測試後，本實驗也進行了初步的應用測試，發現以 E.coli 系統所表達之 HmBRI 和其突變蛋白質，有很高的生物材料應用潛力。 | zh_TW |
| dc.description.abstract | Abstract
Bacteriorhodopsin (BR) is a protein exists in the purple membrane of some archaea and it is expressed when environment is illuminated with strong light or is low in oxygen. BR consists of seven trans-membrane α-helix with a retinal in the core as a chromophore that form schiff base with a Lys residue on the helix G. In the presence of light illumination, protein conformational will change due to the isomerization of retinal, and it further leads to an outward proton transport to generate a proton gradient across membrane. With this chemical gradient, ATP synthase generates energy under low oxygen condition. Just like other photoreceptors, BR features a maximum absorption wavelength and a millisecond time scale photocycle. The fact that BR is highly stable and its relatively high yield in the wild type organism, many scientists regard BR as a one of the best bio-light sensor material for further biotechnology application. The first part of this study focused on characterization of a protein from a gene bop found in Haloarcula marismortui (HmBRI). After sequence analysis to confirm its validity as BR or BR-like protein, the gene was cloned and constructed in E. coli system for over-expression and purification followed by absorbance spectrum measurements, photocycle, and proton pumping activity, this protein was confirmed as a proton pumping bacteriorhodopsin, and it was named HmBRI. The second part of this study mainly dedicated on protein yield test and a carefully evaluation of the potential in biotechnology usage. Various mutants were constructed and it was found that a D94N mutation leads to very high expression level in E.coli expression system and it is stable when treated with heat, light, and even in a almost water-free environment. A series of light intensity experiments unveil a plausible strategy to adopt HmBRI as a light intensity or roughly a UV meter. This study therefore confirms HmBRI as a new bacteriorhodopsin protein with light-driven proton pumping capability and it can be engineered for application in biotechnology. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:17:45Z (GMT). No. of bitstreams: 1 ntu-98-R96b47411-1.pdf: 3181357 bytes, checksum: f70da455411f615f3d25471f5eaa308b (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄
謝誌 II 目錄 III 圖目錄 VI 表目錄 VIII 中文摘要 IX ABSTRACT X 第一章 緒論 1 第一節 Haloarcula marismortui 1 第二節 微生物視紫紅質 (Microbial rhodopsin) 2 第三節 細菌視紫紅質 (BR) 運輸機制 5 第四節 細菌視紫紅質 (BR) 性質研究 10 第五節 細菌視紫紅質 (BR) 應用國內外研究情況 11 5.1 紫膜 (Purple membrane) 11 5.2 BR 與 Purple membrane 之應用 12 5.2.1 光色材料 ( photochromatic material ) 12 5.2.2記憶裝置 (information storage system) 12 5.2.3 其他應用 12 第六節 實驗動機與目的 13 6.1 HmBRI 膜蛋白質大量表現，分離純化 13 6.2 功能鑑定 13 6.3 HmBRI 膜蛋白質可能應用性之研究 13 6.4 HmBRI 蛋白質結晶篩選 13 第二章 材料與方法 14 第一節 HmBRI 大量表現以及純化 14 1.1 HmBRI及其突變蛋白質質體建立 14 1.1.1 質體建構 14 1.1.2 建構 HmBRI 突變蛋白質之方法 14 1.1.2.1. 二階段 PCR 14 1.1.2.2 改良 error-prone PCR：由 Taq 聚合酶進行實驗時產生的突變蛋白質 15 1.2 重組之 HmBRI 和 HmBRI 突變蛋白質之大量表現 15 1.2.1菌種 15 1.2.2 在 E.coli 系統內之蛋白質大量表現 16 1.3 HmBRI 和 HmBRI 突變蛋白質純化與濃縮 16 1.3.1 一般純化流程 16 1.3.1.1 細胞裂解，蛋白質粗萃取 16 1.3.1.2 使用超高速離心獲得細胞膜部分 17 1.3.1.3 使用介面活性劑置換雙層膜中的膜蛋白質 17 1.3.1.4 親合管柱純化 17 1.3.1.5 濃縮 18 1.3.2 修改之蛋白質純化方法 18 第二節 HmBRI 功能測試 19 2.1 蛋白質電泳以及西方點墨法測定 19 2.1.1 SDS-PAGE 蛋白質電泳檢定 19 2.1.2 膠體染色 19 2.1.3 膠體轉印 19 2.1.4 西方點墨免疫呈色 20 2.2 光譜測定 20 2.3 氫離子幫浦測試 20 2.4 光週期測試 20 2.4.1 儀器設置 21 2.4.2 光週期量測 21 第三節 蛋白質結晶 22 3.1 坐式液滴 (sitting drop) 結晶條件篩選 22 3.2 Lipid cubic phase ( LCP ) 結晶法 22 3.3 二維結晶 23 第四節 儀器及設備 24 4.1 核酸電泳設備 24 4.2 蛋白質電泳及轉印設備 24 4.3 離心機 24 4.4 其他 24 第三章 結果與討論 26 第一節 膜蛋白大量表現，分離純化 26 1.1 表現蛋白質之菌體顏色及電泳分析 26 1.2 熱純化與普通純化比較 27 1.3 溫度對 HmBRI 光驅動氫離子幫浦能力測試 29 第二節 HmBRI 與其突變蛋白質之功能測定與應用評估 31 2.1 HmBRI，HmBRI D94N 之比較 32 2.1.1 大量表現於E.coli C43(DE3) 32 2.1.2 吸收光譜，光週期，以及氫離子幫浦測試 33 2.2 Color tuning 36 第三節 細菌視紫質的應用 40 3.1 蛋白質濃縮保存 40 3.2 感光應用構想 41 3.2.1 HmBRI D94N 蛋白質應用 42 3.2.2 HmBRI D94N 全菌應用 43 3.2.3 二維蛋白質結晶實驗 44 第四節 結晶條件篩選 46 4.1 HmBRI 46 4.2 HmBRI D94N 47 第四章 總結 50 第五章 未來展望 51 參考資料 53 附錄：碩士學位口試問與答 57 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細菌視紫質應用 | zh_TW |
| dc.subject | 細菌視紫質 | zh_TW |
| dc.subject | bacteriorhodopsin application | en |
| dc.subject | bacteriorhodopsin | en |
| dc.title | 以生物科技生產Haloarcula marismortui HmBRI蛋白質在生物工業上應用之研究 | zh_TW |
| dc.title | A study on the biotechnical applications of bioengineered Haloarcula marismortui HmBRI | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賴美津,黃慶璨,許瑞祥,黃青真 | |
| dc.subject.keyword | 細菌視紫質,細菌視紫質應用, | zh_TW |
| dc.subject.keyword | bacteriorhodopsin,bacteriorhodopsin application, | en |
| dc.relation.page | 59 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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