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研究生:徐慎行
研究生(外文):Shen Hsing Hsu
論文名稱:酵母菌表達綠豆質子運輸無機焦磷酸水解酵素之純化與定性分析
論文名稱(外文):Expression, Purification, and Characterization of Mung Bean His-tagged Proton-Pumping Inorganic Pyrophosphatase in Yeast
指導教授:潘榮隆潘榮隆引用關係
指導教授(外文):Rong Long Pan
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
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:46
中文關鍵詞:液泡質子無機焦磷酸水解酶蛋白質純化組胺酸標記自體吸收光譜自體螢光光譜
外文關鍵詞:Vacuolar proton pumping pyrophosphataseProtein PurificationHistidine-TagIntrinsic Absorption SpectraIntrinsic Fluorescence Spectra
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液泡質子傳遞無機焦磷酸水解酵素是一個位於植物液泡膜上的蛋白質,主要存在於高等植物和一些細菌中,主要的功能是利用焦磷酸水解產生的能量來將質子由細胞質傳送到液泡中,以產生質子梯度,使液泡維持在酸性的環境中。將蛋白質融合六個連續的組胺酸,使蛋白質可以方便的利用金屬親合性管柱來進行純化,而且利用這個方法可以得到更多且更純的蛋白質,以利蛋白質特性的分析以及結構的探討。在本研究中,我們利用酵母菌將蛋白質表達出來,使用金屬親合性層析管柱(Ni2+-NTA)以及高效率液相層析儀(FPLC)將蛋白質純化出來。利用十二月旨鈉-聚丙烯醯胺凝膠電泳法(SPDS-PAGE)和西方轉漬(Western blot)的分析,知道可以得到純度高、均質的液泡無機焦磷酸水解蛋白,並測得其分子量約為73 kDa.
我們將液泡無機焦磷酸水解蛋白質做一些定性的分析,並且比較野生型的、融合連續六個組胺酸、和純化後的無機焦磷酸水解蛋白質的特性,發現這些不同形式的蛋白質都在其受質(Mg2+/PPi)比例等於1.0且pH 值為8的時候,有最大的酵素活性。另外,離子如鈉離子、氟離子、以及鈣離子等,對於這三種型態的蛋白質都有相同的抑制效果。綜合以上的結果,我們發現純化後的蛋白質和原本的蛋白質其性質是相似的,這結果證明我們這個含有六個組胺酸的無機焦磷酸水解蛋白之異體表現以及純化的方法是可行的。然而,在鉀離子激活酵素活性方面,鉀離子可以激發野生型酵素的活性約10倍,對於有融合連續六個組胺酸的酵素可以激活2-3倍的酵素活性,顯示組胺酸融合的區域可能影響鉀離子的調控。另外利用自身吸收(intrinsic absorption)以及自身螢光(intrinsic fluorescence)光譜的結果得到初步的結果證明當蛋白質和鎂離子、鈣離子結合後會有構型上的改變,未來我們將利用雙圓旋光儀(circular dichroism)進一步研究這些離子的結合機制。
Vacuolar proton inorganic pyrophosphatase (V-PPase ; EC 3.6.1.1) is a membrane-bound protein, found primarily in higher plants and several bacteria. V-PPase can use the outlay of PPi hydrolysis to generate pH gradient across the tonoplast membrane to maintain the vacuoles in acidic condition. The His-tagged fusion protein makes it possible to purify the membrane-bound protein from the membrane by using the DDM (n-Dodecyl β-D-maltoside) as the detergent. With the application of affinity column Ni2+-NTA and FPLC system to purify the V-PPase, analysis by SDS-PAGE and Western blot reveal that the molecular weight of V-PPase is about 73 kDa.
In this studies, we compared the enzymatic activities of wild type membrane-bound V-PPase (mbWTVPP), His-tagged membrane-bound V-PPase (mbHisVPP), and purified His-tagged V-PPase (sHisVPP). The KM of mbWTVPP, mbHisVPP, and sHisVPP were 71.85 μM, 115.04 μM, and 250μM, respectively. The Vmax of these three types of V-PPase are 38.02 μmol PPi mg-1 h-1, 72.46 μmol PPi mg-1 h-1, 307.73 μmol PPi mg-1 h-1, respectively. The maximum enzymatic activity was measurement with the substrate of Mg2+/PPi ratio of 1:1 at pH 8.0. Furthermore, Na+, Ca2+, and F- inhibited the enzyme activities of these three types of V-PPase to the same level. These data showed that the purified His-tagged V-PPase was similar to the membrane-bound V-PPase. However, enzymatic activity of mbWTVPP was stimulated by about 10-fold; however, those of the mbHisVPP and sHisVPP were by about 2-3 fold. Obviously, the His-tagged fusion protein of V-PPase could modify the effect of K+ stimulation. Moreover, spectral studies indicate Mg2+ and Ca2+ could lead to the conformational change of the V-PPase. In further studies, we will use the Circular Dichroism (CD) to observe the secondary structure of V-PPase.
Introduction……………………………………………………………….…...1


Materials and Methods………………………………..…….……….…….…..8


Results……………………………..……………………….………….…..….14


Discussion……………………………..………………...…..……….……….18


References……………………………….………………….….………….….21


Figures and Tables……………………………………………..…………..….27
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