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研究生:蔡昇樺
研究生(外文):Sheng-Hua Tsai
論文名稱:分子伴護子(molecularchaperones)對人類酸性纖維母細胞生長因子(hFGF-1)在摺疊動力學上的影響
指導教授:余靖余靖引用關係
指導教授(外文):Chin Yu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:118
中文關鍵詞:分子伴護子摺疊路徑
外文關鍵詞:molecular chaperonefolding pathway
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蛋白質是一個巨大的分子,需摺疊成固定的三度空間結構才具有活性,近年來,人類發現於細胞中有所謂的分子伴護子(molecular claperone)的存在,這些分子伴護子具有協助其他蛋白質摺疊成正確結構的功能,因此了解其作用的機制就成為了解蛋白質摺疊的一塊墊腳石,於本論文中,選用了本實驗室研究得很詳盡的hFGF-1來和目前了解最透徹的分子伴護子-GroEL進行作用。
由螢光實驗發現只加GroEL會使hFGF-1不穩定,但再加了ATP後使得此不穩定的情況消失,推論這是GroEL先和hFGF-1結合,再加了ATP後又放開,FPLC(gel filtration)及胰蛋白酶裂解(Trypsin digestion)的實驗結果支持這項推論,ANS結合(binding)實驗顯示GroEL在部份開散時效用增強的現象並非來自於疏水性囊袋(hydrophobic pocket)的產生。於動力學上的研究,停止流(Stopped-Flow)螢光實驗的結果顯示,在pH=5.5而且GroEL及ATP存在的情況下,摺疊的速度會加快,抑制流(Quench-Flow) NMR的結果也和此現象相符,而且在比較過有、無GroEL存在的結果發現,GroEL可降低hFGF-1摺疊反應的活化能,使得摺疊路徑產生相當大的改變。

摘要---------------------------------------------------------------------------------1
第一章 簡介---------------------------------------------------------------------2
1.1 蛋白質的摺疊----------------------------------------------------------------2
1.1.1蛋白質摺疊的問題----------------------------------------------------2
1.1.2研究蛋白質摺疊的重要性-------------------------------------------4
1.2 分子伴護子(Molecular Chaperone)--------------------------------------5
1.2.1分子伴護子的定義----------------------------------------------------5
1.2.2分子伴護子的分類----------------------------------------------------6
1.2.3 GroEL-GroES分子伴護子-------------------------------------------8
1.2.3.1 GroEL-----------------------------------------------------------8
1.2.3.2 GroES----------------------------------------------------------12
1.2.3.3 GroEL-GroES與蛋白質摺疊的關係---------------------13
1.3 hFGF-1------------------------------------------------------------------------15
1.4 Quench-Flow NMR---------------------------------------------------------17
第二章 材料與方法------------------------------------------------------------21
2.1 研究蛋白質基本技術-----------------------------------------------------21
2.1.1 Luria Broth培養液(簡稱LB medium) 之配製-----------------21
2.1.2 LB瓊脂培養皿之製備---------------------------------------------21
2.1.3 Glycerol Stock的製作(細菌菌株的保存方法) -----------------22
2.1.4 磷酸緩衝溶液(phosphate buffer)之配製-----------------------22
2.1.5 Resuspension buffer(再懸浮緩衝液)之配製--------------------22
2.1.6 利用高壓均質機取得大腸桿菌中的蛋白質-------------------23
2.1.7 蛋白質的定量-Bradford method (protein assay) --------------24
2.1.8 蛋白質電泳技術(SDS-PAGE) -----------------------------------25
2.2 GroEL、GroES蛋白質的取得---------------------------------------------30
2.2.1 GroEL、GroES之大量表現----------------------------------------31
2.2.2 GroEL蛋白質的純化-----------------------------------------------32
2.2.2.1 蛋白質上清液預濃縮---------------------------------------33
2.2.2.2 利用FPLC純化GroEL-------------------------------------33
2.2.3 GroES蛋白質的純化-----------------------------------------------34
2.2.4 GroEL、GroES蛋白質液濃縮-------------------------------------36
2.3 GroEL及GroES蛋白質的鑑定------------------------------------------37
2.3.1 ESI-Mass的分子量鑑定-------------------------------------------37
2.3.2 CD光譜儀(圓二色光譜儀) ---------------------------------------38
2.4 hFGF-1蛋白質之取得-----------------------------------------------------39
2.4.1 hFGF-1之大量表現-------------------------------------------------39
2.4.2 hFGF-1之純化-------------------------------------------------------40
2.4.2.1親和性肝素(heparin)層析管柱的製備--------------------40
2.4.2.2 以親和性肝素管柱來對hFGF-1進行純化-------------42
2.4.3 hFGF-1流析液之去鹽及濃縮-------------------------------------44
2.4.4 hFGF-1的定量分析-------------------------------------------------45
2.4.5 hFGF-1的螢光性質-------------------------------------------------46
2.5 GroEL-GroES和hFGF-1螢光實驗--------------------------------------48
2.5.1 尿素開散實驗------------------------------------------------------------------49
2.5.2 溫度開散實驗-------------------------------------------------------50
2.5.3 GroEL在部份開散時對hFGF-1的影響-------------------------53
2.5.4 GroEL的ANS尿素開散實驗-------------------------------------54
2.6 GroEL和hFGF-1結合的實驗--------------------------------------------54
2.6.1 FPLC實驗------------------------------------------------------------54
2.6.2 胰蛋白酶裂解(Trypsin digestion)實驗-------------------------56
2.7 動力學實驗-----------------------------------------------------------------57
2.7.1 停止流螢光實驗(Stopped-Flow Fluorescence)---------------57
2.7.2 抑制流(Quench-Flow)NMR實驗-------------------------------59
2.7.2.1 15N-labeled hFGF-1之取得---------------------------------59
2.7.2.2抑制流(Quench-Flow)NMR實驗-------------------------63
第三章 結果與討論-----------------------------------------------------------65
3.1蛋白質的表現與純化------------------------------------------------------65
3.1.1 hFGF-1蛋白質的表現與純化-------------------------------------65
3.1.2 GroEL蛋白質的純化-----------------------------------------------67
3.1.3 GroES蛋白質的純化-----------------------------------------------68
3.2蛋白質的鑑定---------------------------------------------------------------69
3.2.1 GroEL蛋白質的鑑定-----------------------------------------------69
3.2.2 GroES蛋白質的鑑定-----------------------------------------------72
3.3螢光實驗---------------------------------------------------------------------74
3.3.1 GroEL及GroES的尿素開散實驗--------------------------------74
3.3.2 GroEL/GroES對hFGF-1影響的螢光實驗----------------------76
3.3.2.1 尿素變性實驗------------------------------------------------77
3.3.2.2 熱開散實驗---------------------------------------------------81
3.4 GroEL和thFG的結合(binding)實驗------------------------------------84
3.4.1 FPLC實驗------------------------------------------------------------84
3.4.2 胰蛋白酶裂解(Trypsin digestion)實驗-------------------------86
3.5 GroEL的性質探討---------------------------------------------------------88
3.5.1 GroEL的變性實驗--------------------------------------------------88
3.5.2 GroEL在partially unfold時的活性測試-------------------------89
3.5.3 GroEL與ANS的結合(binding)實驗-----------------------------90
3.6 動力學方面的研究--------------------------------------------------------92
3.6.1 停止流(Stopped-Flow)螢光實驗--------------------------------92
3.6.2抑制流(Quench-Flow)NMR實驗--------------------------------95
3.7 未來的展望----------------------------------------------------------------106
第四章 結論--------------------------------------------------------------------107
參考文獻------------------------------------------------------------------------108
附錄------------------------------------------------------------------------------115

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