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論文中文名稱:利用溶菌蛋白酶進行結晶方法的探討 [以論文名稱查詢館藏系統]
論文英文名稱:Comparison of different crystallization techniques testing on lysozyme [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:98
出版年度:99
中文姓名:施惠玲
英文姓名:Hui-Ling Shih
研究生學號:96688003
學位類別:碩士
語文別:中文
口試日期:2010-06-04
論文頁數:79
指導教授中文名:段葉芳;陳銘凱
指導教授英文名:YEH-FANG DUANN;Ming-Kai Chern
口試委員中文名:蔡麗珠;徐駿森
口試委員英文名:Lichu Chi;Chun-Hua Hsu
中文關鍵詞:蛋白質結晶高效能蛋白質養晶技術機器手臂系統
英文關鍵詞:Protein crystallizationHigh-throughput protein crystallization robotic systemsHigh success crystallization screen.
論文中文摘要:在結構生物學領域裡面得到蛋白質結晶,目前在蛋白質結晶學領域裡仍是一個很大的瓶頸,能突破此瓶頸就能得到好的結構。
本論文以溶菌蛋白酶(Lysozyme)作為養晶蛋白質,分別用座式、懸式、微批量三種不同長晶方法與不同養晶條件方式,來長晶並記錄比較其情況與差異。結果在各種養晶方法與條件中,溶菌蛋白酶最佳結晶溫度是室溫、最佳結晶 pH是Na(OAc) pH 4.5與最佳長晶濃度為30 mg/ml中。在微批量養晶方法所需的蛋白質濃度最低,沉澱劑濃度也最低。懸式與座式的結晶差異度很小(因兩者均為藉蒸氣擴散達到過飽和規則排列而結晶出來)。微批量養晶方法的優點是用較低濃度蛋白質與沉澱劑即可養出晶體,若蛋白質容易沈澱或蛋白質無法濃縮至高濃度時,很適合此養晶方法。蛋白質結晶長得太快晶體太小顆,微批量養晶法也是個不錯的改善方法。蒸氣擴散養晶法的優點則是操作容易、簡便、蛋白質晶體撈上X-ray收繞射數據也較容易操作、長晶的速度較快,實驗有時間的限制性或蛋白質量充足時蒸氣擴散養晶法是個較好的選擇養晶方式。
論文英文摘要:In the field of structural biology, protein crystallization remains a bottleneck; therefore, it is important to break through this problem in order to solve protein crystallographic structures. In this study, three different crystallization techniques, i.e. sitting, hanging and microbatch, and different crystallization conditions were applied to crystallize lysozyme and their results were compared. As a result, the best condition for the lysozyme crystal is at room temperature, crystallization reagent of Na(OAc), pH 4.5 and protein concentration of 30 mg/ml. The required concentrations of protein and reagents were lowest in the microbatch method. If protein is easy to precipitate or is not able to concentrate to the higher concentration, microbatch will be the better choice. In addition, if protein crystal grows too fast or is too small, it is better to use the microbatch method. In contrast, there are not many differences between the hanging and sitting methods, because both are vapour diffusion methods. The advantages of vapour diffusion are easy to set up, convenient and easy to mount crystals onto X-ray machine. Also, crystallization is faster in the vapour diffusion technique. If there is a time constraint for experiment or protein amount is ample, the vapour diffusion method will be a better method.
論文目次:目 錄
中文摘要..................................................................................................................i
英文摘要..................................................................................................................ii
誌謝..........................................................................................................................iv
目錄..........................................................................................................................v
表目錄.....................................................................................................................viii
圖目錄.....................................................................................................................ix
中英文縮寫對照表................................................................................................ xi
第一章 緒論 .........................................................................................................1
1.1 溶菌蛋白酶(Lysozyme) .......................................................................1
1.2 蛋白質結晶...........................................................................................4
1.2.1 影響蛋白質結構因素................................................................4
1.2.2 影響蛋白質結晶的因素............................................................7
1.3 養晶原理與結晶方法介紹...................................................................14
1.3.1 結晶的過程................................................................................14
1.3.2 蛋白質結晶方法介紹................................................................16
1.3.2.1 懸式蒸氣擴散養晶方法(Hanging vapor
diffusion method) ..................................................................17
1.3.2.2 座式蒸氣擴散養晶方法(Sitting vapor
diffusion method) ..................................................................18
1.3.2.3 微批量養晶方法(Microbatch under oil method) ...................19
1.3.2.3.1微批量養晶方法用兩種不同比重的油................................19
1.3.2.3.2微批量養晶方法用單種比重的油........................................21
1.3.2.4 毛細管逆擴散養晶方法(Capillary counter-
diffusion method) ..................................................................22
1.3.2.5 微透析養晶法(Microdialysis methods) ..................................24
1.3.2.6 微毛細管結晶系統養晶方法[The plug-based
nanovolume Microcapillary Protein Crystallization
System (MPCS)]......................................................................26
1.4 蛋白質機器養晶原理介紹....................................................................28
1.5 X-ray結晶實驗簡易流程圖..................................................................29
1.6 蛋白質結晶成功率統計分析.................................................................30
第二章 材料與方法.................................................................................................33
2.1 實驗儀器................................................................................................33
2.2.1 自動蛋白質養晶機器...........................................................................34
2.2 蛋白質結晶觀察....................................................................................35
2.3 實驗材料................................................................................................37
2.3.1 耗材與相關藥品.......................................................................37
2.3.2 實驗材料 (蛋白質) .................................................................39
2.4 養晶操作方法的介紹............................................................................44
2.4.1 懸式蒸氣擴散養晶方法(Hanging vapor diffusion) ....................44
2-4.2 座式蒸氣擴散養晶方法 (Sitting vapor diffusion) .....................45
2-4.3 微批量養晶法(Microbatch under oil) ..........................................48
2.5 實驗步驟.................................................................................................49
2.5.1 溶菌蛋白酶最佳化結晶濃度.......................................................50
2.5.2 溶菌蛋白酶主沉澱劑濃度變化....................................................50
2.5.3 溶菌蛋白酶不同溫度變化............................................................50
2.5.4 溶菌蛋白酶酮濃度不同主沉澱濟變化........................................51
2.5.5 溶菌蛋白酶不同觀察時間............................................................51
2.5.6 溶菌蛋白酶pH變化.....................................................................52

2.5.7 溶菌蛋白酶比較不同96養晶條件在不同養晶方法裡
蛋白質結晶造成的差異與結果...................................................52
2.5.8 溶菌蛋白酶用相同96養晶條件在不同養晶方法對蛋白
質結晶造成的差異與結果..........................................................53
2.5.9 將溶菌蛋白酶晶體撈上X-ray收X-ray繞射圖.............................54
第三章 結果.............................................................................................................55
3.1 蛋白質結晶品質的判定依據....................................................................55
3.2 溶菌蛋白酶(Lysozyme)最佳化結晶濃度.................................................58
3.3 主沈澱劑濃度變化....................................................................................59
3.4 溫度變化....................................................................................................60
3.5 同濃度不同主沈澱劑變化........................................................................61
3.6 不同觀察時間............................................................................................63
3.7 pH變化.......................................................................................................64
3.8 比較不同96養晶條件在不同養晶方法裡蛋白質結晶造成的
差異與結果...............................................................................................66
3.9 相同96養晶條件不同養晶方法對蛋白質結晶造成的差異與
結果 .......................................................................................................69
3.9.1 座式(Sitting ).............................................................................69
3.9.2 懸式(Hanging)............................................................................70
3.9.3 微批量(Microbatch) ........................................................................71
3.10 X-ray晶體繞射圖結果 .............................................................................72
第四章 討論.............................................................................................................75
參考文獻...................................................................................................................77
表目錄
表1.1 影響蛋白質結晶幾個重要條件的濃度範圍................................................12
表2.1 不同96養晶篩選條件明細............................................................................40
圖目錄
圖1.1 溶菌蛋白酶一級結構圖..............................................................................2
圖1.2溶菌蛋白酶(lysozyme)蛋白質結構圖.........................................................3
圖 1.3 dynamic light scattering (DLS) ...................................................................5
圖 1.4 圖示說明瞭解蛋白質折疊狀態的NMR Spectroscopy方法.....................6
圖 1.5 SDS PAGE圖...............................................................................................7
圖1.6測試蛋白質養晶濃度試劑 [Pre-Crystallization Test (PCT)] ....................8
圖1.7 影響蛋白質結晶的幾個重要條件..............................................................11
圖1.8 概要說明結晶的過程 ................................................................................15
圖 1.9 圖解說明懸式蒸氣擴散的養晶方法.........................................................17
圖 1.10 圖解說明座式蒸氣擴散養晶方法...........................................................18
圖1.11 微批量養晶法用兩種不同油(Microbatch under oil) 的圖解說 明................................................................................................................20
圖1.12微批量養晶法用單種油(Microbatch under oil) 的圖解說
明................................................................................................................21
圖1.13圖解說明毛細管逆擴散法(Capillary counter-diffusion methods)
所使用的相關設備與實驗成果介紹.......................................................23
圖1.14圖解說明微透析法(Microdialysis)的蛋白質養晶方法所使用的
相關設備與實驗成果介紹.......................................................................24
圖1.15圖解說明微毛細管蛋白質結晶系統MPCS (Microcapillary Protein Crystallization System)養晶方法所使用的相關設備與實驗成果介紹.................................................................................................................26
圖1.16 TargetDB 實驗結果所得的統計圖.............................................................31
圖1.17 TargetDB生物有機體的來源所佔分佈比例圖.........................................32
圖2.1自動養晶機器(Gilson 925 PC Workstation Crystallization
System)設備介 紹.........................................................................................34
圖2.2觀察蛋白質結晶時所用的顯微鏡與恆溫箱.................................................35
圖2.3溶菌蛋白酶蛋白質純度SDS電泳圖............................................................40
圖2.4 圖解說明懸式蒸氣擴散養晶方法及其相關設備介紹.................................44
圖2.5圖解說明座式蒸氣擴散養晶方法及其相關設備介紹..................................47
圖2.6圖解說明自動養晶機器微批量(Microbatch)養晶方法及其相
關設備介紹.....................................................................................................48
圖3.1 介紹各種不同好壞結晶相.............................................................................57
圖3.2不同蛋白質濃度對蛋白質結晶的影響..........................................................58
圖3.3不同沈澱劑濃度對蛋白質結晶造成的影響與差異......................................59
圖3.4不同溫度對蛋白質結晶造成的影響與差異..................................................60
圖3.5同濃度不同沈澱劑對蛋白質結晶造成的影響與差異..................................62
圖3.6不同時間對蛋白質結晶造成的影響與差異..................................................63
圖3.7不同pH對蛋白質結晶造成的影響與差異...................................................65
圖3.8 不同養晶方法對蛋白質結晶造成的差異.....................................................68
圖3.9 座式蒸氣擴散養晶方法第二天長出完整的晶體與放置50天
後晶體依然保存完整的樣子.........................................................................69
圖3.10 懸式蒸氣擴散養晶方法第二天長出完整的晶體與放置50天
後晶體長相差異比較...................................................................................70
圖3.11微批量養晶法(Microbatch)同樣96養晶條件有3種不同現象
結果產生.......................................................................................................71
圖3.12 溶菌蛋白酶晶體X-ray晶體繞射圖……....................................................72
圖 3.13 溶菌蛋白酶大顆與小顆晶體的X-ray繞射圖比較.....................................73
圖 3.14 溶菌蛋白酶晶體在不同pH下X-ray繞射圖結果........................................74
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論文全文使用權限:同意授權於2014-07-09起公開