蛋白質特定方向固定化-以α-amylase為例;+C2640 Site-specific Immobilization of α-amylase Protein

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Title:	蛋白質特定方向固定化-以α-amylase為例;+C2640 Site-specific Immobilization of α-amylase Protein
Authors:	曾冠維;Kuan-Wei Tseng
Contributors:	化學工程與材料工程研究所
Keywords:	物理吸附;特定方向;固定化;分子嵌合;physical adsorption;site-specific;immobilization;docking
Date:	2008-07-04
Issue Date:	2009-09-21 12:30:09 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	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。 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.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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