Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3346
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dc.contributor.advisor林松池zh_TW
dc.contributor.advisorSung-Chyr Linen_US
dc.contributor.author蔡松原zh_TW
dc.contributor.authorTsai, Sung-Yuanen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T05:31:47Z-
dc.date.available2014-06-06T05:31:47Z-
dc.identifier.urihttp://hdl.handle.net/11455/3346-
dc.description.abstractAbstract Recent advances in genomics and proteomics have led to a need for efficient methods for the purification of recombinant proteins. Among the many purification techniques, immobilized metal affinity chromatography(IMAC) exhibits unique characteristics for the one-step purification of poly(His)-tagged proteins and has thus become a routine purification technique in research and is gaining significance in industrial applications. To facilitate the application of IMAC in industrial processes, it is necessary to understand the complicate interactions between protein molecules and the adsorbents. In this study, a systematic study aiming at understanding the chromatographic behaviors of four poly(His)-tagged recombinant proteins, possessing different molecular weights and numbers of subunits, was carried out in both native and denaturing conditions. Four adsorption models, Langmuir, Scatchard, Temkin, and Langmuir-Freundlich, were used to evaluate the major adsorption parameters. It was confirmed in this study that the adsorption parameters were strongly dependent on the sizes and the numbers of subunits of the proteins. The results obtained in this study provide crucial information for elucidating the chromatographic behaviors of proteins and are instrumental in the simulation, scale-up, and operation of industrial chromatographic processes.en_US
dc.description.abstract中文摘要 近年來在純化基因重組蛋白質的技術中,固定化金屬親和層析技術是最被廣泛應用的技術,且開始受到工業界的重視。具有聚組織胺標籤的基因重組蛋白質可經由固定化金屬親和層析技術達到單一步驟純化與固定化的目的。要有效進行層析製程的設計、最適化條件及放大生產必須全面性的了解固定化金屬與蛋白質間的各種相互關係。本研究以系統性的定性及定量方法與理論來解釋說明這些相互關係。本研究利用分別具有不同聚組織胺標籤數目與分子量的四個標的蛋白質(epimerase、N-carbamoylase、D-hydantoinase和racemase),探討原始狀態及變性狀態之蛋白質於商業膠體及合成之配位體密度膠體的吸附行為。我們利用四個恆溫吸附模式分析實驗數據,即Langmuir模式、Scatchard 模式、Temkin模式和Langmuir- Freundlich模式,來計算吸附係數。實驗證明,改變吸附材的金屬離子密度,蛋白質大小及聚組織胺標籤數目,都是影響蛋白質吸附量的重要因素。利用恆溫吸附模式得到的吸附係數,可說明合作關係吸附程度及非均相的吸附行為。本研究成果可幫助了解基因重組蛋白質與固定化金屬離子的相互關係,並且作為設計放大固定化金屬親和層析技術製程的重要指標。zh_TW
dc.description.tableofcontents目錄 頁次 中文摘要.....................................................Ⅰ 英文摘要.....................................................Ⅱ 第一章 緒論..................................................1 第二章 文獻回顧..............................................2 2-1 蛋白質純化……………………………………… 2 2-1-1 酵素之介紹…………………………………2 2-1-2 蛋白質純化……………………………………3 2-1-3 純化策略……………………………….……3 2-2 固定化金屬親和層析法(immobilized metal-ion affinity chromatography)之介紹………………………………………7 2-2-1 固定化金屬親和層析法……………………7 2-2-2 常用之金屬離子………………………………8 2-2-3 金屬螯合劑……………………………………8 2-2-4 吸附載體……………………………………10 2-3 影響吸附與脫附之因素……………………………………10 2-3-1 螯合劑結構及金屬離………………………11 2-3-2 移動相:pH值,緩衝液和離子強度……11 2-3-3 配位體密度…….…………………………15 2-3-4 添加劑和蛋白質取代劑(protein displacers).......................22 2-4 恆溫吸附研究……………………………………………22 2-5 標的酵素介紹……………………………………………25 2-5-1 N-acetyl D-glucosamine 2-epimerase (epimerase)之簡介……………………25 2-5-2 D-hydantoinase之簡介…………………27 2-5-3 N-carbamoyl-L-amino acid amidohydrolase (N-carbamoylase)之簡 介................................29 2-5-4 N-Acylamino acid racemase(Racemase) 之簡介............................30 第三章 試驗裝置與藥品……………………………………………………33 3-1 試驗裝置…………………………………………………33 3-2 藥品………………………………………………………33 第四章 試驗方法………………………………………………………35 4-1 四個酵素的培養、純化及收集…………………………35 4-1-1 LB固態洋菜培養基之製備………………35 4-1-2 LB液態培養基之製備………………………35 4-1-3 epimerase菌種培養………………………35 4-1-4 N-carbamoylase菌種培養………………36 4-1-5 D-hydantoinase菌種培養………………37 4-1-6 Racemase菌種培養…………………………37 4-1-7 酵素收集及純化………………………38 4-1-7-1 native 狀態的酵素純化……38 4-7-1-2 denaturing狀態的酵素純化…39 4-2 蛋白質分析…………………………………………………42 4-2-1 蛋白質濃度分析…………………………42 4-2-2 SDS-PAGE分析……………………………42 4-3 膠體合成…………………………………………………43 4-3-1 各種配位體密度之膠體合成………………43 4-3-2 金屬離子之鍵結……………………………44 4-3-3 銅離子鍵結量量測…………………………45 4-4 標的蛋白質對Ni-NTA之吸附………………………………45 4-4-1 Ni-NTA膠體的蛋白質吸附…………………45 4-4-2 Ni-NTA膠體鎳離子鍵結量量測……………45 4-5 標的蛋白質對蛋白質膠體之吸附....................46 4-5-1 各種比例配位體密度膠體之吸附…………46 4-5-2 恆溫平衡吸附實驗…………………………47 4-5-2-1 native蛋白質之恆溫吸附實 驗........................47 4-5-2-2 denaturing蛋白質之恆溫 吸附實驗..................47 第五章 結果與討論…………………………………………… 49 5-1 四個標的蛋白質之性質……………………………………49 5-2 酵素於Ni-NTA膠體之吸脫附情形…………………………50 5-3 配位體密度蛋白質膠體銅離子鍵結量……………………54 5-4 四個標的於配位體密度膠體之吸附情形…………………54 5-5 恆溫吸附試驗……………………………………………55 5-5-1 native狀態之蛋白質吸附…………………55 5-5-2 denaturing狀態之蛋白質吸附…………64 第六章 結論……………………………………………………… 70 第七章 附錄……………………………………………………… 72 第八章 參考文獻………………………………………………… 73zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系zh_TW
dc.subjectisotherms adsorptionen_US
dc.subject恆溫吸附zh_TW
dc.subjectrecombinant proteinsen_US
dc.subjectimmobilized metal ion affinity chromatographyen_US
dc.subject基因重組蛋白質zh_TW
dc.subject固定化金屬親和層析法zh_TW
dc.titleAdsorption behaviors of recombinant proteins on immobilized metal affinity adsorbents.en_US
dc.title基因重組蛋白質在金屬螫合層析吸附材的吸附行為zh_TW
dc.typeThesis and Dissertationzh_TW
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