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研究生:鄭銘松
研究生(外文):Ming-Sinng Cheng
論文名稱:生長激素摺疊之研究-似一階相變反應模型
論文名稱(外文):Study of Growth Hormones Folding: First-order-like Phase Transition Model
指導教授:甘魯生
指導教授(外文):Lou-Sing Kan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
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:59
中文關鍵詞:蛋白質摺疊圓二色旋光光譜儀動態光散射儀摺疊中間物似一階相變反應模型
外文關鍵詞:circular dichroism spectroscopy (CD)dynamic light scattering (DLS)quasi-static-like processfirst-order-like phasetransitionprotein folding
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蛋白質摺疊可自然發生但是卻容易發生錯誤摺疊 (misfold) 、形成聚合體 (aggregate) 而不具生物活性功能。本研究中,使用純化自豬的腦下垂體生長激素當作實驗材料,所得的結構特性可以用來作為基因重組香魚 (Plecoglossus altivlis) 及石斑魚 (Epinephelus awoara) 生長激素結構之參考,並利用 quasi-static-like processes來恢復生長激素的結構及活性功能。為了解生長激素構形的改變及分子大小分佈 (particle-size-distribution, PSD) ,使用圓二色旋光光譜儀 (circular dichroism spectroscopy, CD) 及動態光散射儀 (dynamic light scattering, DLS) 來監測。由圓二色旋光光譜儀實驗結果發現在蛋白質摺疊過程中,蛋白質二級結構於第一階段摺疊反應後即開始就出現了,而其受熱變性曲線在第四摺疊態後,方呈現協同效應之 S 型圖形,顯示蛋白質三級結構在第四摺疊態後方形成,推論 quasi-static-like processes 方法可使三種的生長激素摺疊成具有正確結構的蛋白質;並推論蛋白質二級結構可能是扮演幫助蛋白質摺疊的重要角色。這些穩定平衡的狀態的摺疊中間物 (M1到M4) 在當時的緩衝溶液條件中是處於接近自然態位能的最低位能狀態,並推測在一般情況之下,重摺疊含有變性劑的蛋白質是一個 first-order-like phasetransition。

The folding of a protein may be a spontaneous process and independent of its folding path but it may misfold and aggregate. Thus, we designed a stepwise quasi-static process to restore the growth hormone structures and to change refolding potentials of the solvents in a gradual manner in the hope that conformational changes of the associated intermediates, Mi (i = 1 to n), between U and N, might be detected by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS). Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions within the protein were restored during the last two refolding stages, as elucidated by thermal stability tests. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrunk to native-like-size after the first refolding stage. Our observations suggest that stable protein refolding intermediates can be obtained by using quasi-static TED processes. Namely, an unfolded protein which refolds back through a carefully designed stepwise quasi-static-like process may experience a gradual change in its environment and thus will not be trapped in a “burst phase” or form aggregates.

目次 頁次
中文摘要………………………………………..……………….. 5
英文摘要………………………………………..…….…………..6
縮寫表…………………………………………..…….…………..7
第一章 緒論
§1-1 前言
1-1.1 蛋白質摺疊………………………….………………..9
1-1.2 相轉換………………………………………………..11
§1-2 生長激素
1-2.1簡介…………………………………………………...12
1-2.2蛋白質結構…………………………………………...14
1-2.3生理功能……………………………………………...14
§1-3 基因工程重組蛋白質……………………..……….…17
§1-4 研究目標…………………………………..……….…19
第二章 實驗分析儀器與實驗原理
§2-1 紫外光譜分析原理與應用
2-1.1紫外光譜分析原理……………………………..……..21
2-1.2 Beer’s law與蛋白質濃度的測定……….…………….23
§2-2 圓二色光譜分析原理與應用
2-2.1圓二色旋光光譜儀分析蛋白質原理……..…………..23
2-2.2 SELCON3分析蛋白質結構之原理………...………..25
§2-3 動態光散射儀原理與應用……………………….……27
§2-4 細胞增生分析原理與應用………………………..…...29
第三章 實驗方法及步驟
§3-1 Quasi-static-like TED process 緩衝溶液………….....31
§3-2 表現基因工程重組香魚、石斑魚生長激素………...31
§3-3 內含體的純化………………………………………...32
§3-4 生長激素的變性與展開……………………………...32
§3-5 利用 quasi-static-like TED process 將生長激素重新重摺疊…………………………………………….………....32
§3-6 利用圓二色旋光光譜儀分析蛋白質重摺疊中間物二級結構……………………………………………..…….....33
§3-7 利用動態光散射儀分析蛋白質重摺疊中間物分子大小分佈情形…………………………………………..……….34
§3-8 細胞增生實驗分析……………………………………34
第四章 實驗結果與討論
§4-1 利用圓二色旋光光譜儀檢測摺疊中間物……..……..36
§4-2 細胞增生實驗結果……………………….……………36
§4-3 利用動態光散射儀分析蛋白質重摺疊中間物分子大小分佈情形………………………………………………………37
第五章 實驗討論
§5-1 利用類似靜態熱力學動態平衡透析方法來重摺疊生長激素並利用圓二色旋光光譜儀檢測摺疊中間物…………39
§5-2 類似靜止熱力學動態平衡透析方法可以避免蛋白重摺疊時發生聚集………………………………………..……...40
§5-3 基因工程重組香魚、石斑魚生長激素的生物活性具有種特異性……………………………………………..………..42
§5-4 甘露醣在蛋白質摺疊時是扮演化學輔佐子的角色….43
§5-5 蛋白質摺疊符合 collapse and sequential model….…..43
§5-6 含有變性劑的重摺疊方法是一個 first-order-like phase transition process…………………………….….……..45
§5-7 Quasi-static-like TED process可以廣泛地適用於其他蛋白質………………………………………….…………..47
§5-8 生物體內蛋白質的摺疊可能是 continuous transition path………………………….…………………….…..47
第六章 結論……………………….……………………….…..49
儀器與藥品……………………………………………………..53
參考文獻………………………………………………………..56

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