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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉懷勝 | |
| dc.contributor.author | Ming-Jun Peng | en |
| dc.contributor.author | 彭明鈞 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:53:03Z | - |
| dc.date.available | 2005-07-30 | |
| dc.date.copyright | 2005-07-30 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-28 | |
| dc.identifier.citation | Aceto, A., Dragani, B., Allocati, N., Masulli, M., Petruzzelli, R. and DiIlio, C.,
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35443 | - |
| dc.description.abstract | 摘要
本實驗以直接稀釋法進行長時間蛋白質復性，探討復性液組成變化對於蛋白質摺疊結構及聚集體生成的影響。運用分析方法包括活性測定及280 nm的吸收值測量，直接或間接計算各種狀態之蛋白質量。並使用穿透式電子顯微鏡觀察蛋白質的聚集形態，及Congo Red染色法討論蛋白質摺疊結構，藉此瞭解蛋白質摺疊路徑中分子內作用力與分子間作用力之關係。 實驗中首先以Tris-HCl緩衝液，或加入1.2mM GSH，或加入1.2mM GSSG進行稀釋法復性，觀察復性液中各成份影響蛋白質摺疊，發現於Tris-HCl緩衝液和加入GSH環境下復性的蛋白質，分子間作用力為主導蛋白質摺疊主要因素，而在加入GSSG環境下復性時，分子內與分子間作用力產生相互競爭。其次調整復性液中GSH與GSSG之組成比例(固定總濃度2[GSSG]+[GSH]=3.6mM)，控制A280/A260比例為0.1~0.3，且添加低濃度尿素進行長時間(六天)蛋白質復性，結果發現低濃度尿素可延緩聚集體形成的速率，並於不同時間下形成各類的聚集體﹔添加2M尿素可完全抑制聚集體生成，使反應逐漸朝原態結構方向反應；當復性液中A280/A260比值為0.3（即[GSSG]/[GSH]=1.2mM/1.2mM）時，蛋白質活性回收率由65%(復性四小時)逐漸增加為96%(復性六天後)，並由比較結果得知復性液中添加GSH可增加10%左右之活性回收率。 | zh_TW |
| dc.description.abstract | Abstract
This study involved the investigations of lysozyme refolding and aggregates refolding by direct dilution. The renaturation of active protein and aggregate was determined by activity measurement, absorbance at 280nm, together with Congo Red binding assay, ThT fluorescence, TEM and optical measurement. To understand the competition behavion between the protein refolding and aggregation, we investigated the effect of different constituents of renaturation buffer on refolding performance by direct dilution method. Experiments results showed that low concentration urea addition in the renaturation process could suppress protein aggregation to stimulate the pathway of correct refolding at high protein concentration. Chang [2004] proved that the maximum refolding efficiency occurred in 60 minute with renaturation buffer while maintaining [GSSG]/[GSH]≧1 the ratio A280/A260 above 0.3 for various protein concentrations. Subsequently, the refolding experiments of renaturation buffer with various combination of [GSSG]/[GSH] and urea concentration were carried out for 6 days. The results showed that activity recovery increased gradually from 65% to 96% by 10-fold dilution at the concentration 1.2mM/1.2mM [GSSG]/[GSH] and 2M Urea of renaturation buffer during 6day incubation. | en |
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| dc.description.tableofcontents | 中文摘要……………………………………………………………………………..Ⅰ
英文摘要……………………………………………………………………………..Ⅱ 目錄…………………………………………………………………………………..Ⅲ 圖目錄………………………………………………………………………………..Ⅵ 表目錄…………….…………………………...…….……………………………….III 第一章 緒論…………………………………………………………………………..1 第二章 文獻回顧……………………………………………………………………..2 2-1 溶菌酶（Lysozyme）簡介………………………………………………..2 2-1-1 溶菌酶之介紹…………………………………………………...2 2-1-2 溶菌酶的構造…………………………………………………...3 2-1-3 溶菌酶活性測定原理…………………………………………...5 2-2 蛋白質結構與穩定蛋白質分子之作用力……….……………………..6 2-3 蛋白質之變性…………………………………………………………...7 2-4 蛋白質的復性程序………………………………………………….....11 2-4-1降低變性劑的濃度……………………………………………...11 2-4-2移除變性劑……………………………………………………...14 2-4-3加入添加劑……………………………………………………...17 2-5 蛋白質復性動力學…………………………….……………………....19 2-6 化學變因與物理變因對溶菌酶復性的影響…………………….…....22 2-6-1化學變因的影響………...............................................................22 2-6-2物理變因的影響………………………………………………...27 2-6-3溶菌酶之摺疊…………………………………………………...29 2-7 榖胱甘肽（Glutathione）介紹…………………………………………..32 2-8 類澱粉沉積症（amyloidosis）…………………………………….........35 2-8-1 Congo Red 染色法…………......................................................38 2-8-2 Thioflavin T（ThT）螢光分析……………………………….......39 第三章 實驗裝置、藥品與步驟……………………………………………………..40 3-1 實驗裝置……………………………….………………........................40 3-2 藥品…………………………….………………………........................40 3-3 實驗步驟………………………………………………….....................42 3-3-1 溶菌酶之活性測定………………….…………........................42 3-3-2 溶菌酶之變性……………………………………………….....42 3-3-3 溶菌酶之直接稀釋法復性………………………….................43 3-3-4 Congo Red 染色法.................................……………..............44 3-3-5 Thioflavin T (ThT)螢光測試………………………………....45 3-3-6 穿透式電子顯微鏡……………………….................................45 第四章 結果與討論…………………........................................................................46 4-1 討論原態與失活溶菌酶於波長280nm之吸收值……........................46 4-2 直接稀釋法復性之定量實驗流程…………………….........................48 4-3 失活與復性蛋白質溶液中觀察所形成的聚集現象….........................51 4-3-1 Congo Red鍵結失活溶菌酶於分光光譜儀中分析.................51 4-3-2失活溶菌酶於穿透式電子顯微鏡（TEM）負染下觀察………..56 4-3-3失活溶菌酶於Thioflavin T（ThT）螢光分析…………………...58 4-3-4 以外觀及光學顯微鏡觀察聚集體…………………….............59 4-4 添加尿素於復性緩衝液進行直接稀釋法復性….................................62 4-4-1 添加尿素於Tris-HCl緩衝液………………………………......62 4-4-2 添加尿素於GSH與GSSG之復性緩衝液………………….....69 4-5 以不同比例之GSSG/GSH復性緩衝液並添加尿素進行復性………81 4-6 以定量及Congo Red染色法討論失活蛋白質摺疊途徑…………….98 4-7 觀察聚集體於復性過程之改變……………………………………...101 4-7-1 利用波長450nm測量聚集體之濁度變化…………………...101 4-7-2 聚集體A*於系統是否影響後續聚集體生成與活性表現…..103 第五章 結論………………………………………………………………………..106 參考文獻……………………………………………………………………………109 英文縮寫對照表……………………………………………………………………115 附錄(A)……………………………………………………………………………...116 A-1 溶菌酶(Lysozyme)之活性測定…………….......................................116 A-2 溶菌酶之吸收值測定………………………………………………..118 附錄(B)……………………………………………………………………………...120 B-1 GSH、GSSG、EDTA、Urea於A280與A260吸收值比較……………..120 B-2 GSH與GSSG隨時間放置濃度變化情形………............................121 圖目錄 圖2-1-1 溶菌酶(Lysozyme)之一級結構……………………………………………..3 圖2-1-2 溶菌酶分子結構圖………………………..……………………………..…4 圖2-1-3 溶菌酶催化水解微生物細胞璧多醣類之間的β-（1→4）-glycosidic 鍵結……………………………………………………………………………6 圖2-2 尿素與胍鹽酸破壞蛋白質分子結構之機制……………………..………10 圖2-3 GSSG/GSH與蛋白質的反應簡示圖……………………………..………12 圖2-4 連續式透析法實驗裝置圖……………………………..…………………13 圖2-5 中空纖維薄膜法實驗裝置圖……………………………………………..13 圖2-6 SEC復性圖，依照分子大小將失活蛋白質、聚集體、再摺疊蛋白、變 性劑分別析出……………………………………………………………14 圖2-7 層析結果圖………………………………………………………………..15 圖2-8 逆微胞法之復性機制圖………………………………………..…………16 圖2-9 不同尿素濃度對蛋白質聚集反應之影響…………………….………….17 圖2-10 蛋白質分子摺疊過程中，可能朝向正確摺疊路徑，亦可能朝向因多胜肽 鏈間之互相作用所造成的聚集反應……………………………..………19 圖2-11 以動力學描述正確摺疊與聚集現象之競爭性反應…………………......20 圖2-12-1 A260與GSH及GSSG濃度之關係圖………………………………….23 圖2-12-2 A280與GSH及GSSG濃度之關係圖……………………………….…23 圖2-13-1 各種不同初始濃度的失活溶菌酶稀釋10倍的活性回收率[GSSG]/[GSH]作圖………………………………………………………………………25 圖2-13-2 各種不同初始濃度的失活溶菌酶稀釋10倍的活性回收率對A280/A260 作圖……………………………………………………………………....25 圖2-13-3 初始濃度3g/L的失活溶菌酶分別稀釋10倍、20倍、30倍及50倍的活性回收率對A280/A260作圖……………………………………………26 圖2-13-4 初始濃度3g/L的失活溶菌酶分別稀釋10倍、20倍、30倍及50倍的 活性回收率對[GSSG]/[GSH]作圖……………………………………...26 圖2-14 不同溫度下利用稀釋法的復性結果……………………………………28 圖2-15 失活溶菌酶可能摺疊之路徑…………………………………………....30 圖2-16 溶菌酶摺疊過程中，復性動力學與雙硫鍵鍵結之相關性…………….31 圖2-17-1 還原態榖胱甘肽(GSH)之構造圖………………………………………..33 圖2-17-2 氧化態榖胱甘肽(GSSG)之構造圖…………………………………...….33 圖2-18 類澱粉纖維結構………………………………………………......……..35 圖2-19-1 Congo Red之化學結構……………………………………………..…..38 圖2-19-2 Thioflavin T之化學結構……………………………….…………….…39 圖4-1 以波長280nm測量原態蛋白質與完全失活六小時蛋白質於濃度0.1~0.6g/L之吸收值……………………………………………………47 圖4-2 以直接稀釋法復性蛋白質之實驗流程圖及定量方法………….….…49 圖4-3-1 Congo Red染劑鍵結於原態及失活蛋白質，於全波長400~700nm下掃描觀察550nm處之第二波形…………………………………………..52 圖4-3-2 Congo Red染劑鍵結於原態及失活蛋白質並扣除溶液中對照組………………………………………………………………………..53 圖4-3-3 簡單蛋白質摺疊路徑與聚集反應圖……………..…………………….55 圖4-4 利用TEM負染方法觀察失活蛋白質與染劑析出物…………...……..57 圖4-5-1 失活溶菌酶（0.5g/L）於ThT螢光分析……………………..………..58 圖4-5-2 不可溶性聚集體外觀以及光學顯微鏡放大400倍下觀察………..…..60 圖4-6-1 以Tris-HCl緩衝液(不含尿素、不含GSSG/GSH)進行直接稀釋法復對蛋白質定量作圖………………………………………………………..63 圖4-6-2 以Tris-HCl緩衝液添加0.5M尿素(不含GSSG/GSH)進行直接稀釋法復性對蛋白質定量作圖……………………………………………..…65 圖4-6-3 以Tris-HCl緩衝液添加2M尿素(不含GSSG/GSH)進行直接稀釋法復性對蛋白質定量作圖…………………………………………………..65 圖4-6-4 以Tris-HCl緩衝液(不含尿素、不含GSSG/GSH)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………..66 圖4-6-5 以Tris-HCl緩衝液添加0.5M尿素(不含GSSG/GSH)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………….…..68 圖4-6-6 以Tris-HCl緩衝液添加2M尿素(不含GSSG/GSH)以直接稀釋法復性對染劑C.R鍵結分析……………………………………………..…….68 圖4-6-7 以1.2mM GSH復性液(不含尿素、GSSG)進行直接稀釋法復性對蛋白質定量作圖………………………………………………………..……69 圖4-6-8 以1.2mM GSH復性液添加0.5M尿素(不含GSSG)進行直接稀釋法復性對蛋白質定量作圖………………………………………………..…70 圖4-6-9 以1.2mM GSH復性液添加2M尿素(不含GSSG)進行直接稀釋法復性對蛋白質定量作圖………………………………………………..……70 圖4-6-10 以1.2mM GSH復性液(不含尿素、GSSG)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………………...71 圖4-6-11 以1.2mM GSH復性液添加0.5M尿素(不含GSSG）進行直接稀釋法復性對染劑C.R鍵結分析……………………………………………...72 圖4-6-12 以1.2mM GSH復性液添加2M尿素(不含GSSG)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………..….72 圖4-6-13 以1.2mM GSSG復性液(不含尿素、GSH))進行直接稀釋法復性對蛋白質定量作圖…………………………………………………………..73 圖4-6-14 以1.2mM GSSG復性液添加0.5M尿素(不含GSH)進行直接稀釋法復性對蛋白質定量作圖…………………………………………………..74 圖4-6-15 以1.2mM GSSG復性液添加2M尿素(不含GSH)進行直接稀釋法復性對蛋白質定量作圖…………………………………………………......74 圖4-6-16 以1.2mM GSSG復性液(不含尿素、GSH))進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………….……75 圖4-6-17 以1.2mM GSSG復性液添加0.5M尿素(不含GSH)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………..76 圖4-6-18 以1.2mM GSSG復性液添加2M尿素(不含GSH)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………..76 圖4-7 失活蛋白質的再摺疊途徑……………………………………………..77 圖4-8 失活溶菌酶以直接稀釋法復性，於不同復性劑成份(不添加尿素)之摺疊路徑圖………………………………………………….………….…79 圖4-9 失活溶菌酶以直接稀釋法復性，於不同復性劑成份並添加2M尿素之 摺疊路徑圖…………………………………………………………...80 圖4-10-1 復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對蛋白質定量作圖………………………………………………………………...…….81 圖4-10-2 添加0.5M尿素於復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對蛋白質定量作圖……………………………………………...………..….83 圖4-10-3 添加2M尿素於復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對蛋白質定量作圖…………………………………………………………..83 圖4-10-4 復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………………………….….84 圖4-10-5 添加0.5M尿素於復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………………..85 圖4-10-6 添加2M尿素於復性緩衝液(A280/A260=0.1)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………………...85 圖4-10-7 復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對蛋白質定量作 圖……………………………………………………………………..…86 圖4-10-8 添加0.5M尿素於復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對蛋白質定量作圖…………………………………………………………..87 圖4-10-9 添加2M尿素於復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對蛋白質定量作圖………………..……………………………………..…87 圖4-10-10 復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………………………..…88 圖4-10-11 添加0.5M尿素於復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對染劑C.R鍵結分析…………………………………………………….89 圖4-10-12 添加2M尿素於復性緩衝液(A280/A260=0.2)進行直接稀釋法復性對染劑C.R鍵結分析………………………………………………………89 圖4-10-13 復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對蛋白質定量作圖……………………………………………………………………....90 圖4-10-14 添加0.5M尿素於復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對 蛋白質定量作圖……………..……………………………………..…91 圖4-10-15 添加2M尿素於復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對蛋 白質定量作圖………..………………………………………………..91 圖4-10-16 復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對於染劑C.R鍵結析………………………………………………………………….….93 圖4-10-17 添加0.5M尿素於復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對染劑Congo Red 鍵結分析……………………………………...……94 圖4-10-18 添加2M尿素於復性緩衝液(A280/A260=0.3)進行直接稀釋法復性對染 劑Congo Red 鍵結分析……………………………..…………….…94 圖4-11 復性液組成比例對活性回收率影響 (a)比較各復性液比例A280/A260之活性回收率………………….……...97 (b)比較各復性液比例A280/A260並添加0.5M尿素之活性回收率………………………………………………………………….…...97 (c)比較各復性液比例A280/A260並添加1M尿素之活性回收率……....97 (d)比較各復性液比例A280/A260並添加2M尿素之活性回收率…….…97 圖4-12 失活蛋白質於復性時各種情況之摺疊路徑……………………..........100 圖4-13 波長450nm測量聚集體之濁度變化 (a)復性液比例A280/A260=0.1及添加不同濃度尿素於復性四小時內之濁度變化……………………………………………………..……………101 (b)復性液比例A280/A260=0.1及添加不同濃度尿素於復性六天內之濁度變化……………………………………………………… …… ………101 (c)復性液比例A280/A260=0.2及添加不同濃度尿素於復性四小時內之濁度變化………….…………………………………………………….…102 (d)復性液比例A280/A260=0.2及添加不同濃度尿素於復性六天內之濁度變化……………………………………………………………………..102 (e)復性液比例A280/A260=0.3及添加不同濃度尿素於復性四小時內之濁度變化……………………………………………………………….….102 (f)復性液比例A280/A260=0.3及添加不同濃度尿素於復性六天內之濁度變化………………………………………………………………….….102 圖4-14-1 不同復性液組成比例及尿素添加下，復性四小時後離心與未離心聚集體(A*)對總聚集體(At)量生成的影響…………………………………104 圖4-14-2 不同復性液組成比例及尿素添加下，復性四小時後離心與未離心聚集體(A*)對活性回收率的影響………………………………………..…105 表目錄 表2-1 各種蛋白質變性方法………………….……………….……………………..8 表2-2 觀察聚集體之各種分析方法……………………………………..…………21 表2-3 酸鹼值對復性的影響………………………………………………………..27 表2-4 不同溫度下利用稀釋法的復性結果………………………………………..28 表2-5 蛋白質構形改變與相關疾病發生……….………………………………….36 表2-6 測量類澱粉纖維的方法..................................................................................37 表4-1 表列實驗復性(含變性)過程中觀察到的各種聚集體……………..…….…59 表4-2不同的復性液成份（不添加尿素），復性六天後，各蛋白質形態之百分比…………………………………………………………...………………79 表4-3 不同的復性液成份並添加2M尿素，復性六天後，各蛋白質形態之百分比…………………………………………………………………………....80 表4-4 比較2M尿素復性液當中含有1.2mM GSH /1.2mM GSSG及1.2mM GSSG其活性回收率（%）之差異…………………………….…………96 表4-5 不同的復性劑組成以及添加尿素（0M、2M），復性於六天後，各蛋白質形態之百分比與波形強度值………………………………………………98 | |
| dc.language.iso | zh-TW | |
| dc.subject | 變性 | zh_TW |
| dc.subject | 復性 | zh_TW |
| dc.subject | 溶菌酶 | zh_TW |
| dc.subject | Denaturation | en |
| dc.subject | Renaturation | en |
| dc.subject | Lysozyme | en |
| dc.title | 復性緩衝液的組成對蛋白質摺疊與聚集體生成之影響 | zh_TW |
| dc.title | The effect of different constituents of renaturation buffer on refolding performance and aggregate formation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王勝仕,侯劭毅 | |
| dc.subject.keyword | 溶菌酶,復性,變性, | zh_TW |
| dc.subject.keyword | Lysozyme,Renaturation,Denaturation, | en |
| dc.relation.page | 122 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-28 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| 顯示於系所單位： | 化學工程學系 | |
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