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標題: 基因重組蛋白質在氫氧基磷灰石金屬螯合親和層析吸附材的吸附行為
Adsorption behaviors of recombinant proteins on hydroxyapatite-based immobilized metal affinity chromatography adsorbents
作者: 林芃幟
Lin, Peng-Jhih
關鍵字: Hydroxyapatite
Adsorption isotherm
Protein purification
出版社: 化學工程學系所
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摘要: 本研究應用氫氧基磷灰石(Hydroxyapatite)為固定化金屬離子親和吸附材,並以Fe3+為配位金屬離子。並將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完成純化的目的,再深入地了解蛋白質間與固定化金屬的各種相互關係的應用。本研究探討原始狀態(native conditions)及變性狀態(denaturing conditions)之蛋白質在氫氧基磷灰石膠體的吸附行為,分別利用具有不同聚組織胺標籤數目與分子量大小的五個標的蛋白質(esterase、epimerase、D-hydantoinase、N-carbamoylase和racemase)。再利用四個恆溫吸附模式進行分析實驗數據,即Langmuir模式、Langmuir-Freundlich模式、Temkin模式和Scatchard模式,計算吸附係數qm、Kd與n值。實驗證明,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。由實驗的數據結果得到分子量越大,其qm吸附量越少;聚組織胺標籤數目越多,其kd值越小,表示親和性越大。從恆溫吸附模式可以幫助說明吸附的程度與協同關係之吸附行為,並且可以作為設計放大固定化金屬親和層析技術製程的重要指標。
The effects of molecular weight and number of poly(His)-tags on the adsorption of recombinant proteins on a hydroxyapatite-based immobilized metal affinity adsorbent were investigated in this study. Under native conditions, the adsorption isotherms were well-fitted by the Langmuir-Freundlich isotherm model. The maximum adsorption capacity was found to decrease with the increase in molecular weight. The binding affinity increased significantly with the number of poly(His)-tags. The adsorption capacities for esterase and epimerase were exceptionally high due to probably multilayer adsorption. Under denaturing condition, both the Langmuir and the Langmuir-Freundlich isotherm models were found to well-fitted the adsorption behavior of all five proteins studied. Under denaturing conditions, the binding affinities for all five proteins studied were essential identical and were much lower than that under native conditions, indicating that the Fe(III)-charged hydroxyapatite may not be appropriate for protein purification.
其他識別: U0005-2508200711011000
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