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研究生:翁如潔
研究生(外文):Ju-Chieh Wung
論文名稱:人類APP和BACE蛋白質之表現與純化
論文名稱(外文):Overexpression and Purification of Human APP and BACE
指導教授:孔繁璐孔繁璐引用關係
指導教授(外文):Fan-Lu Kung
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
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:68
中文關鍵詞:蛋白質純化蛋白質表現
外文關鍵詞:APPpurificationBACEoverexpression
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阿茲海默氏症(Alzheimer’s disease)是一種漸進式的神經退化性疾病。這種疾病主要的病理特徵為在腦部皮質和海馬迴存在著老年斑(amyloid plaque)和神經纖維糾結(neurofibrillary tangle)。老年斑主要由Abeta蛋白質塊構成,Abeta是由amyloid precursor protein (APP)經由beta-和gamma-secretase依序切割所形成的。科學家發現beta-secretase為一種新的酵素,而將其命名為beta-site APP cleaving enzyme (BACE)。為了要研究BACE如何催化APP的水解反應,取得大量的APP和BACE蛋白質是首要的條件。為了要取得BACE的受質,我們建構了一個包含glutathione S-transferase (GST)融合APP695基因的質體,利用此質體在大腸桿菌內表現出GST-APP695融合蛋白後,以親合性層析法來純化此蛋白。依據相同的原理,我們將全長的BACE基因轉型至大腸桿菌內,並且大量表現GST-BACE融合蛋白,發現這個蛋白質是以inclusion bodies的形式存在。我們嘗試將inclusion bodies經denaturation/refolding等方法處理以取得具有酵素活性的BACE。此外,為了要瞭解N-glycosylation對BACE活性的影響,我們將全長的BACE基因轉殖至秋行軍蟲細胞中,大量表現並得到具有酵素活性的N-glycosylated BACE。我們利用以上的方法來建立大量表現和純化APP及BACE的實驗流程,期望能夠幫助我們達成最終的實驗目標。我們最終的實驗目標是利用體外酵素動力學研究的方法來探討BACE和APP之間辨識的特異性。
Alzheimer’s disease is a neurodegenerative disorder of which one of the major pathological features is the presence of amyloid plaque and neurofibrillary tangle in brain cortex and hippocampus. Amyloid plaque is mostly composed of Abeta, which is generated from proteolytic cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase in sequential. A novel protein designated as beta-site APP cleaving enzyme (BACE) contains all the known characteristics of beta-secretase. To investigate how BACE catalyzes the proteolytic reaction of APP, it is essential to obtain large amounts of APP and BACE. To obtain the substrate of BACE, a construct encoding glutathion S-transferase and APP695 was used to overexpress the GST-APP695 fusion protein in Escherichia coli, which was later purified using affinity chromatography. The plasmid containing the full length BACE gene was transformed into E. coli and used to overexpress GST-BACE as GST-APP695. It was found that the fusion protein formed inclusion bodies when being overexpressed. It was attempted to develop a denaturation/refolding scheme to obtain active BACE from inclusion bodies. To assess the effects of N-glycosylated on BACE activity, the full length BACE gene was transfected into Spodoptera frugiperda cells to obtain N-glycosylated active BACE. Hopefully an overexpression/purification scheme can be established to obtain large quantities of APP and BACE efficiently to investigate the substrate recognition of BACE using in vitro kinetics studies.
Chinese Abstract… … … … … … … … … … … … … …ii
English Abstract… … … … … … … … … … … … … iii
Abbreviations… … … … … … … … … … … … … … …iv
Introduction… … … … … … … … … … … … … … … 1
Objective… … … … … … … … … … … … … … … … 6
Materials and Methods… … … … … … … … … … … … 8
A. Strategies to purify recombinant proteins… … … … 8
B. Materials… … … … … … … … … … … … … … … 8
C. Procedures… … … … … … … … … … … … … … …9
Construction of expression plasmids… … … … … … … 9
Protein purification methods in E. coli… … … … … … 12
Protein purification in baculovirus system… … … ..… 16
Results… … … … … … … … … … … … … … … … …21
A. Construction of expression plasmids… … … … … … 21
B. Protein expression in E. coli… … … … … … … … 23
C. Purification of proteins expressed in E. coli… … … 26
D. protein expression in baculovirus system… … … … …29
Discussion… … … … … … … … … … … … … … … …32
Figures… … … … … … … … … … … … … … … … …40
Tables… … … … … … … … … … … … … … … … … 61
Reference… … … … … … … … … … … … … … … … 64
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