- 學位論文
由內涵體經由三相分離重新摺疊與純化重組之鏈霉抗生物素蛋白
Simultaneous refolding and purification by three-phase partitioning of recombinant Streptavidin from inclusion bodies
摘要
為開發快速、高效率分離和純化蛋白質。三相分離法（Three Phase Partioning,TPP）作為蛋白質純化的先導步驟，日益受到重視。其方法是加入硫酸銨鹽類、緩衝液與有機醇混合，以獲得三相分離，其三相分離分別為有機相、界面沉澱與水相。從界面沉澱分離出的蛋白質可以用選定的緩衝液回溶為高濃度的較純蛋白質溶液。TPP研究重點在於不同鹽濃度、有機溶劑、pH值與各種蛋白質的交互作用。我們研究三相分離(TPP)，以純化後的內涵體溶解並正確摺疊具有活性之蛋白質。在這項研究中，從內涵體回收重組鏈霉抗生物素蛋白分離通過使用三相（TPP）分離過後。該方法為粗萃液：三級丁醇1：1（重量/體積），5％硫酸銨，pH 8.0(Tris Buffer)中為37℃溫度下，得到最高蛋白質的purification（fold）（3.67）和yield（％）（164.5％）。基於本研究結果，可以進一步改良鏈霉抗生物素蛋白的純化方法，以利於檢測，標記和純化的產業應用。
並列摘要
In recent years, the trend has been developed rapid, efficient and scale up for separation and purification of protein . Three-phase partitioning ,a protein purification approach, is carried out by mixing ammonium sulfate,buffer and organic solvents to obtain partitioned organic phase, interfacial precipitate and aqueous phase. The effects of different salt concentration, organic solvents, and pHs of various protein has been the focus of investigation. The purpose of this thesis is to apply three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. In this study, the recovery recombinant streptavidin, an important bio-conjugation protein for various biotechnology applications, were isolated from inclusion bodies by using three-phase partitioning (TPP). The system consisted of crude enzyme extract : t-butanol 1:1 (w/v),5% ammonia sulfate, pH 8.0(Tris Buffer) with incubation temperature of 37 oC showed the highest protein purification(fold) (3.67) and yield(%) (164.5%). In conclusion , protocol of streptavidin purification can be further developed to Streptavidin ,which can see wide applications in biosensing and preparative biotechnology field .