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標題: Adsorption behaviors of recombinant proteins on immobilized metal affinity adsorbents.
作者: 蔡松原
Tsai, Sung-Yuan
關鍵字: isotherms adsorption;恆溫吸附;recombinant proteins;immobilized metal ion affinity chromatography;基因重組蛋白質;固定化金屬親和層析法
出版社: 化學工程學系
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.

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。
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