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姓名 曾冠維(Kuan-Wei Tseng)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 蛋白質特定方向固定化-以α-amylase為例
(Site-specific Immobilization of α-amylase Protein)
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摘要(中) 蛋白質以隨機的方式固定於固體基材上,常因不適當的位向而使
蛋白質失去部分活性,影響了蛋白質檢測的精確性。而有特定位向固
定化的方法中,如在蛋白質N 端或C 端接上一段短鏈組氨酸與金屬
離子表面專一性的螯合,但這種方法過程複雜、費用高,而且也無法
得到100%的活性。所以在本研究中,我們發展一種以物理吸附且可
以有位向性固定化的方法,此方法是在蛋白質活性位置的相反方向依
蛋白質表面胺基酸的特性及分佈來設計一個結合強度較強的ligand。
在實驗中,目標蛋白為α-amylase from Aspergillus oryzae, 所使用的
ligand 為 3,3’,4,4’ - Biphenyltetracarboxylic dianhydride (BPDA)。利用
分子嵌合的模擬來預測結合位置及結合蛋白質在矽膠體上等溫吸附
的實驗及活性測試來驗證蛋白質的吸附位向。而TAKA 在BPDA 上
的解離常數比隨機吸附的值低,約為0.28~0.76×10-6M,且其生物活
性也比隨機吸附的高,所以證實BPDA 這個基材有效的提升了與蛋
白質的結合強度和特定位向的吸附,以及分子嵌合對結合位置的預測
了解蛋白質的吸附位向。
摘要(英) Random protein immobilization usually suffers from serious loss of
the specific bioactivity of the immobilized protein. Oriented protein
immobilization of histidine tagged protein on metal chelating resin does
not guarantee 100% exposure of the active site. In this study, we develop
a new method for oriented immobilization. Design an affinity ligand
according to the characteristic and distribution of amino acids at the
opposite to the active site. The target protein is α-amylase from
Aspergillus oryzae, and the searched ligand is 3,3’,4,4’ -
Biphenyltetracarboxylic dianhydride (BPDA). We predict the possible
binding sites by using molecular dockikng. And at the experiments, we
attach BPDA to the surface of silica gel and via isotherm adsorb and
bioactivity assay show oriented immobilization owns superior specific
activity than random immobilization.
關鍵字(中) ★ 物理吸附
★ 特定方向
★ 固定化
★ 分子嵌合
關鍵字(英) ★ physical adsorption
★ site-specific
★ immobilization
★ docking
論文目次 摘要................................................................................................................................ I
Abstract ......................................................................................................................... II
誌謝.............................................................................................................................. III
目錄.............................................................................................................................. IV
圖目錄........................................................................................................................ VII
表目錄.......................................................................................................................... IX
第一章 緒論 .............................................................................................................. 1
1.1 研究動機 .............................................................................................................. 1
1.2 研究目的 .............................................................................................................. 2
第二章 文獻回顧 ...................................................................................................... 3
2.1 蛋白質固定化 ...................................................................................................... 3
2.1.1 共價鍵結 (covalent binding) ............................................................................. 3
2.1.2 生物親和性 (bioaffinity) ................................................................................... 5
2.1.2.1 protein A or protein G ........................................................................... 5
2.1.2.2 生物素鍵結(biotin-binding) ................................................................. 6
2.1.2.3 標籤(tags) .............................................................................................. 7
2.1.3 物理吸附 ............................................................................................................ 8
2.1.3.1 等溫吸附曲線 ....................................................................................... 9
2.2 蛋白質的吸附位向 ............................................................................................ 12
2.3 分子嵌合 ............................................................................................................ 14
2.3.1 分子嵌合介紹 .................................................................................................. 14
2.3.2Autodock ............................................................................................................ 15
2.4 澱粉分解酶介紹 ................................................................................................ 18
第三章 實驗設備、藥品、方法 ............................................................................ 20
3.1 實驗藥品 ............................................................................................................ 20
3.2 實驗設備 ............................................................................................................ 21
3.3 實驗方法 ............................................................................................................ 22
3.3.1 基材表面改質 .................................................................................................. 22
3.3.1.1 表面胺基改質 ..................................................................................... 23
3.3.1.2 Ligand 接枝 ........................................................................................ 23
3.3.1.3 琥珀酸酐阻隔(blocking) .................................................................... 23
3.3.2 蛋白質吸附曲線 .............................................................................................. 24
3.3.3 蛋白質活性測定 .............................................................................................. 25
3.3.3.1 測量原理 ............................................................................................. 25
3.3.3.2 操作方法 ............................................................................................. 26
3.3.4 吸附後蛋白質穩定性 ...................................................................................... 26
3.3.5 蛋白質濃度測定-Bradford method ............................................................... 27
3.3.5.1 Bradford 方法 .................................................................................... 27
3.3.5.2 測量方法 ............................................................................................. 27
第四章 結果與討論 ................................................................................................ 28
4.1 Ligand 設計 ....................................................................................................... 28
4.1.1 TAKA 活性位置電荷分析 ......................................................................... 28
4.1.2 苯環對TAKA 的嵌合分析 ........................................................................ 29
4.1.3 Ligand 的設計 ............................................................................................. 31
4.2 分子嵌合 – 預測TAKA 與BPDAcp 的結合位置 ......................................... 33
4.3TAKA 與BPDA 在水相的活性測試 .................................................................. 37
4.4 Ligand 接枝於矽膠體表面 ............................................................................... 38
4.5 TAKA 於不同基材表面的等溫吸附實驗 ........................................................ 38
4.6 蛋白質吸附後活性測試...................................................................................... 41
4.7 TAKA 在不同pH 值及鹽濃度下的穩定度 ....................................................... 42
4.8 TAKA 的吸附位向 ............................................................................................ 43
第五章 結論 ............................................................................................................ 45
參考文獻...................................................................................................................... 47
附錄.............................................................................................................................. 52
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指導教授 阮若屈(Ruoh-Chyu Ruaan) 審核日期 2008-7-23
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