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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.author | Sz-Wei Wu | en |
dc.contributor.author | 吳思緯 | zh_TW |
dc.date.accessioned | 2021-07-01T08:12:56Z | - |
dc.date.available | 2021-07-01T08:12:56Z | - |
dc.date.issued | 2003 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75383 | - |
dc.description.abstract | Sac7d蛋白質是一個在耐熱性古生菌Sulfolobus acidocaldarius發現的小分子帶鹼性的染色體蛋白,Sac7d對於溫度、酸性環境以及化學物質有很高的耐受性,它對DNA序列並沒有特定的專一性,一般認為它的功能是維持Sulfolobus acidocaldarius的染色質在高溫環境中的穩定性。Sac7d是以單體型式存在,我們認為多聚體的Sac7d 應該會表現出與單體Sac7d不同的DNA結合性質。為了得到多聚體的Sac7d,利用蛋白質工程的方法將一段a螺旋的肚肚接在Sac7d的尾端,理論上藉著螺旋—螺旋作用力形成的leucine zipper,兩個或更多的單體Sac7d會因此而結合成一個多聚體。先在電腦模擬下確認此方法的可行性,利用大腸桿菌大量表現此重組蛋白,根據其性質,在加熱後去除大部分的雜蛋白,再藉由陽離子交換管柱層析法純化,即可得到純度95%以上的產物。藉由分子篩管柱層析、化學鏈結實驗、表面物質共振實驗與圓二色光譜分析探討其物理及化學特性。以分子篩管柱層析、化學鏈結實驗、表面物質共振實驗證明水溶液下多聚體存在的事實,圓二色光譜實驗確認此融合蛋白二級結構的組成及其對雙股DNA構型的影響,利用表面物質共振實驗得到其對雙股DNA的動力學數據,從x光晶體燒射實驗得到蛋白質立體結構。此次的設計可以當作一個成功的範例—如何賦予蛋白質額外的功能卻又不會使原來的性質喪失,更進一步的,也許能以此方式得到一個衍生自Sac7d的耐熱性DNA酵素,如同Tag DNA聚合?應用於PCR上,或是甚至於外加上一段穿透細胞膜模組,可以如同膜蛋白一般鑲嵌在微胞上,當成一個高穩定性的DNA載體,應用於基因治療上。 | zh_TW |
dc.description.abstract | The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid and chemical agents. This protein binds DNA without any particular sequence preference. We propose that multimeric Sac7d would show different DNA binding properties. In order to form dimeric Sac7d, a helical peptide was fused after the C-terminal end of Sac7d. Two or more monomers theoretically will interact with each other via hydrophobic force, as in leucine zipper, to form a dimer. Large amount of recombinant Sac7d was obtained by expression in E. Coli and purification by heating and ion-exchange chromatography. The physical and chemical properties of Sac7d were verified by gel-filtration, chemical cross-linking with SDS-PAGE, SPR, and CD. The tertiary structure was determined by X-ray crystallography. This model works as a successful template that endows protein new function without losing original properties. Furthermore, a thermo-stable enzyme that targets DNA, such as Tag DNA polymerase using in PCR, deriving from Sac7d might be practicable, or fusing a transmembrane domain to Sac7d which inlays in micelles works as a highly stable DNA carrier used in gene therapy. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2003 | en |
dc.description.tableofcontents | 目錄 Ⅰ 圖目錄 Ⅴ 表目錄 Ⅷ 英文摘要 Ⅸ 中文摘要 Ⅹ 目次 第一章 序論 1. 引言1 2. DNA結合蛋白 1 3. Sulfolobus acidocaldarius與Sac7d蛋白 4 4. 蛋白質與蛋白質間的作用力 5 5. GCN4與Leucine zipper模組 9 6. 融合蛋白(fusion protein) 10 第二章 材料與方法 材料 11 儀器 13 實驗方法 14 1. 融合蛋白之設計 14 (1) 尋找蛋白質結合模組 14 (2) 電腦模擬 14 2.菌株製備 14 (1) 目標蛋白之嵌入基因(Insert gene)製備 14 (2) 限制?處理(Enzyme cutting) 15 (3) 去除雜質(clean up) 15 (4) 黏合(Ligation) 16 (5) 轉型(Transformation) 16 (6) 確認轉型成功 16 3.菌體保存 16 4.表現條件測定 17 5.耐酸耐熱性質測試 17 6.純化方法 17 (l) 粗抽液製備 17 (2) 加熱 17 (3) 離子交換管柱層析法 18 (4) 蛋白質電泳 18 (5) 透析 18 (6) 冷凍乾燥 19 7.蛋白質分子量鑑定 19 8.圓二色(Circular dichroism , CD)光譜實驗 19 (1) 原理 19 (2) 方法 21 (3) S7d-14Lz與Sac7d CD光譜 21 (4) S7d-14Lz與Sac7d對雙股DNA CD光譜的影響 21 9.化學性鏈結實驗 22 (1) 原理 22 (2) S7d-14Lz與Sac7d之比較 22 (3) S7d-14Lz不同濃度間之比較 23 10. Surface Plasma Resonance (SPR)實驗 23 (1) 原理 23 (2) 方法 26 (3) 數據分析 27 11.分子篩管柱(Gel filtration)分析 27 12.晶體培養 27 13. x光繞射數據收集與處理(data collection and processing) 29 14.繞射數據初步分析 29 15.相位問題(phasing problem) 29 16.分子模型建立與最佳化(model building and refinement) 29 17.結構確認(validation) 31 18.分子結構圖形繪製 31 19.分子間作用力測定 31 20. DNA構形分析 31 第三章 實驗結果 1. S7dLz多聚體之電腦模擬 32 2. S7d-14Lz之純化 32 (1) 加熱 32 (2) 陽離子交換管柱層析 33 (3) 蛋白質電泳 33 (4) 透析 33 (5) 蛋白質分子量鑑定 33 3. 圓二色光譜實驗 33 (l) S7d-14Lz與Sac7d CD光譜之比較 33 (2) S7d-14Lz與Sac7d對雙股DNA CD光譜影響之比較 34 4.化學性鏈結實驗 35 (1) S7d-14Lz與Sac7d之比較 35 (2) S7d-14Lz不同濃度間之比較 35 5. Surface Plasma Resonance (SPR)實驗 35 (l) 折射指示值(RU)變化之比較 35 (2) 動力學分析 35 6.分子篩(Gel filtration)管柱層析分析 36 7.結晶條件 36 8. x光繞射數據收集與處理 37 9.繞射數據初步分析 37 10.相位問題 38 11.分子模型建立與最佳化 38 12.結構確認 38 13. 晶格堆積 38 14. S7d-14Lz之立體結構 39 15. S7d-14Lz單體間作用力(intermolecular interaction) 39 16. DNA與S7d-14Lz作用力 40 17. DNA構形變化 40 第四章 討論 41 第五章 結論與未來展望 45 圖 46 表 99 參考文獻 110 | |
dc.language.iso | zh-TW | |
dc.title | 耐熱性蛋白Sac7d多聚體設計結構之決定 | zh_TW |
dc.title | Design and structure determination of a thermostable multimeric DNA binding protein using Sac7d as a template | en |
dc.date.schoolyear | 91-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 115 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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