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研究生:張學凱
研究生(外文):Hsueh-Kai Chang
論文名稱:復性緩衝液中的成份變化對蛋白質復性之研究
論文名稱(外文):The effect of change in refolding buffer on the refolding efficiency of protein
指導教授:劉懷勝劉懷勝引用關係
指導教授(外文):Hwai-Shen Liu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:109
中文關鍵詞:復性緩衝液溶菌酶核糖核酸酶蛋白質復性GSSG/GSH
外文關鍵詞:lysozymeprotein refoldingrefolding bufferGSSG/GSHRNase A
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本實驗主要是採用直接稀釋法來進行蛋白質的復性研究,並分成變性條件及復性兩部份來探討。前半段我們把重心放在變性時間的長短、變性劑的不同等變因來比較其對後續復性效果的影響。而後半段的實驗則是對不同濃度的蛋白質、不同的稀釋倍率作復性的研究,比較其復性回收率的變化。

由於廖[2003]證實復性緩衝液中[GSSG]/[GSH]的成份比例會隨時間的放置而增加,然而這樣的現象導致了復性效果的明顯提升。本實驗則是以新鮮配製各種不同[GSSG]/[GSH]的比例來模擬復性緩衝液成份隨時間的變化,而蛋白質的復性效率結果相當類似。而在稀釋復性的過程中發現,影響蛋白質復性效果的因素包括復性緩衝液的成份、蛋白質的濃度、稀釋倍率等等。其中復性緩衝液成份比例我們以A280/A260為指標來作觀察。對這些變因的影響結果我們把他整理出一個回歸式: Act(%) = 275N2.15 P-1.94N+1 I-0.14
由此回歸式,我們只要得知復性緩衝液A280/A260(N)、蛋白質濃度(I)及稀釋倍率(P),就可對蛋白質的復性回收率作一概略的預測。

此外,實驗的過程中我們意外的發現復性緩衝液中EDTA的存在與否對蛋白質的復性復性效果有重大影響。當復性緩衝液含EDTA時:1.使GSH氧化成GSSG的速率大幅減緩;2.使蛋白質復性過程糾結體的產生變嚴重。因此,為了得到更好的復性效果,在溶菌酶的復性過程中,如使用A280/A260較高的復性緩衝液時,我們建議添加EDTA,而使用A280/A260較低的復性緩衝液時,則建議不加EDTA較佳。


This study mainly investigates the refolding efficiency of denatured protein using the method of direct dilution. The research is comprised of two major sections.The first section investigates the effect of denaturation period and different denatured agent on protein refolding efficiency. The second section primarily discusses the difference of protein recovery with different concentration of protein and dilution factor.

It is proven that the ratio [GSSG]/[GSH] increases with storage time [Liao,2003], which enhances the protein renaturation. For this reason, [GSSG]/[GSH] freshly prepared to model the previous work and the refolding efficiency obtained is found to be very similar. A ratio of absorbance, A280/A260 is used in this study to quantify the condition of refolding buffer. It is found that the A280/A260 and concentration of protein is a key factor that affects the refolding efficiency. As a result, we propose an empirical correlation to predict protein recovery efficiency:
Act(%) = 275N2.15 P-1.94N+1 I-0.14
From this correlation, we can approximate the recovey of enzyme activity if A280/A260(N), protein concentration(I) and dilution fold(P) are known.

Besides, we discover that the refolding efficiency of protein is different if EDTA is added or not in the refolding buffer. It is found that refolding buffer with EDTA will reduce the oxidation rate of GSH to GSSG and result in a serious protein aggregation. Therefore, it is recommended that EDTA addition is necessary for refolding buffer with a high A280/A260, and it is not necessary for refolding buffer with a low A280/A260.


中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅱ
目錄……………………………………………………………………..Ⅲ
圖目錄…………………………………………………………………..Ⅵ
表目錄…………………………………………………………………..Ⅹ
第一章 緒論……………………………………………………………..1
第二章 文獻回顧………………………………………………………..2
2-1 蛋白質的結構……………………………………………...……..2
2-1-1 穩定蛋白質分子的作用力……………………………….2
2-1-2 蛋白質分子之立體結構………………………………….4
2-2 蛋白質之變性…………………………………………………….7
2-2-1 蛋白質之變性方式……………………………………….7
2-2-2 常見之變性劑…………………………………………….7
2-3 蛋白質之復性…………………………………………………....9
2-3-1 直接降低變性劑濃度…………………………………...11
2-3-2 分離變性劑與蛋白質…………………………………...17
2-3-3 加入添加劑…………………………..………………….23
2-4 蛋白質介紹………………….………………………………….25
2-4-1 溶菌之簡介………………………………………...........25
2-4-2 溶菌酶之構造………..…………………………….……25
2-4-3 溶菌酶之活性測定……………………………………...28
2-4-4 核糖核酸酶之簡介………………………………….......29
2-4-5 核糖核酸酶之活性測定………………………………...32
2-5 穀胱甘肽(Glutathione)………………………………………….34
2-5-1 穀胱甘肽……………………………………………..….34
2-5-2 緩衝液中穀胱甘肽含量之簡易分析………………..….37
第三章 實驗裝置、藥品與步驟………………………………………..40
3-1 實驗裝置、藥品………………………………………………...40
3-1-2 實驗藥品………………………………………………...40
3-2 實驗步驟………………………………………………………...42
3-2-1 溶菌酶之活性測定………………………………….......42
3-2-2 核糖核酸酶A之活性測定………………………………42
3-2-3 溶菌酶、核糖核酸酶A之變性…………………………43
3-2-4 以不同變性條件作變性對溶菌酶復性效果的影響…...43
3-2-5 溶菌酶、核糖核酸酶A之直接稀釋法復性……………..44
第四章 結果與討論……………………………………………………48
4-1 改變變性條件對直接稀釋法效果之影響………………….......48
4-1-1 變性時間長短對溶菌酶復性效果之影響………….......48
4-1-2 改變變性劑成份之影響………………………………...51
4-1-3 變性環境溫度對溶菌酶復性效果之影響……………...54
4-1-4 變性環境pH值對溶菌酶復性效果之影響……………..57
4-2 復性緩衝液成份變化對直接稀釋法復性程序之影響………...59
4-2-1 各種初始濃度失活溶菌酶之直接稀釋復性實驗……...60
4-2-2 同初始濃度失活溶菌酶之直接稀釋復性實驗………...63
4-2-3 溶菌酶復性回收率之回歸式…………………………...67
4-2-4 核糖核酸酶A之直接稀釋復性…………………………76
4-2-5 復性緩衝液中有無EDTA對復性效果之影響…………83
第五章 結論……………………………………………………………92
參考文獻………………………………………………………………..95
附錄A-1 溶菌酶之活性測定……………...………………………....102
附錄A-2 核糖核酸酶之活性測定…...................................................104
附錄 B-1 稀釋復性實驗原始數據…………………………………..105
附錄 B-2 Matlab回歸程式…………………………………………...109



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