Ribonuclease A 於逆微胞系統中的復性及其界面現象之研究

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题名:	Ribonuclease A 於逆微胞系統中的復性及其界面現象之研究
作者:	李雅雯;Yi-Wen Li
贡献者:	化學工程與材料工程研究所
关键词:	電導度;界面;摺疊;復性;逆微胞;核糖核酸酵素;螢光;refolding;AOT;ribonuclease A;percolation
日期:	2001-06-19
上传时间:	2009-09-21 12:19:57 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	逆微胞法復性可解決基因工程中因蛋白質濃度過高形成內聚體(inclusion body)的問題。本研究以AOT與isooctane形成的逆微胞進行Ribonuclease A(RNase A)的復性，深入探討界面層在復性過程中所扮演的角色。首先，探討RNase A溶入逆微胞的特性與穩定性：第一，透過活性量測實驗得知native RNase A在30小時內的活性是穩定的。第二，透過DLS(Dynamic light scatter)量測，native RNase A溶入逆微胞中會使得其粒徑略增，而denatured RNase A本身結構鬆散，粒徑較大，因此溶入會使逆微胞粒徑增加量較native大很多。第三，利用電導度測量不同系統產生percolation現象溫度的差異，含有native RNase A的系統產生percolation的溫度會隨其與逆微胞界面層的靜電作用力而有差別；而denatured RNase A則由於疏水作用力均會提升界面硬度，當RNase A與界面層為靜電吸引力時，效果更明顯。以上的結果顯示添加RNase A對逆微胞的結構與特性會有影響。改變逆微胞的粒徑與水相的pH值，以改變RNase A與界面層間的作用力，進行復性實驗，探討RNase A於逆微胞內的復性，逆微胞分子界面層所扮演的角色。結果發現當RNase A吸附在逆微胞界面層上時，逆微胞的粒徑大小並不會影響復性。且RNase A吸附在逆微胞界面層上時，復性效果較佳，若RNase A與逆微胞分子界面層為靜電斥力或位於水相中，復性效果則較差，因此可說明逆微胞界面層對復性是有相當助益的，並非只提供孤立蛋白質分子的作用。配合螢光光譜實驗說明構形的摺疊所需時間與活性的恢復所需的時間有相當大的落差，可能是因為Tyr之位置不在活性區，所以螢光之的結果並無法直接與活性恢復結果相關。由以上論述可知逆微胞界面層對RNase A的復性過程有幫助，提供蛋白質在較疏水狀態時吸附的界面，以進行正確的構形摺疊，此與chaperone提供疏水界面，使蛋白質吸附以進行構形摺疊的原理是相同的。 A novel process utilized by reverse micelles has been developed to resolve the inclusion body which produced by recombinant technology. Refolding of denatured ribonuclease A (RNase A) used reverse micelles formulated with AOT in isooctane was studied as a model system to explore the mechanism of protein refolding at micellar interface. The activity of native RNase A in reverse micelles is stable within 30 hours by enzyme activity measurement. The data measured by using DLS (dynamic light scattering), it was found that the average diameter of reverse micelles with denatured RNase A were larger than the empty ones, however, for native RNase A in reverse micelles, the effect is minimum. Solubilizing denatured RNase A in reverse micelles make the AOT interface stiff and repress the percolation threshold between reverse micelles by the conductivity measurement. Native RNase A would change the percolation behavior with the surface changes. This result indicated that solubilized RNase A in reverse micelles would interfere the interactions between AOT surfactants and RNase A, and it would change the AOT interface structure. To investigate the role of interface played in refolding, the effects of operational parameters such as Wo(=[H2O]/[AOT]) and pH were examined on refolding. The denatured RNase A adsorpted onto the micellar surface due to hydrophobic and electrostatic interactions. Denatured RNase A adsorpted onto the micellar interface resulting in a better enzyme activity recovery, indicating that micellar interface play an important role for RNase A refolding. From fluorescence measurement, structure refolding can finish immediately and activity recovering need about thirty hours. The mechanism of the micelle-assisted refolding was proposed on the basis of interaction between micellar interface and RNase A. In the present study, we show that the chaperone-like function of reverse micelles can be utilized in refolding of RNase A.
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