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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/6345


    Title: 在模式生物系統中表現異源性膜蛋白次單元體:微粒體甲烷單氧化酵素的B 次單元體;Heterologous Expression of Membrane Protein Subunit in Host Organisms: The Subunit B of pMMO
    Authors: 張廷安;Ting-An Chang
    Contributors: 化學研究所
    Keywords: 銅離子(I)結合蛋白;微粒體甲烷單氧化酵素;銅離子運輸;膜蛋白表現;pMMO;membrane-protein expression;copper trafficking;Cu(I)-binding protein
    Date: 2007-12-19
    Issue Date: 2009-09-22 10:17:58 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 從嗜甲烷菌Methylcoccus capsulatus (Bath)中分離的微粒體甲烷單氧化酵素B次元體,在過去的研究中證實了它與還原態銅離子具有高度的親合力,這個現象是因為蛋白質C端水溶性次區塊所蘊含的九到十二個還原態銅離子所造成,這些銅離子在甲烷催化過程中,扮演著將電子傳進催化中心以活化甲烷的角色。然而,在最近報導的X光結晶結構中,B次單元體與大量還原態銅離子的結合情形並未得到證實;可能是因為這些銅離子容易在高氧濃度下或者嚴苛的純化過程中流失。為了要再度確認微粒體甲烷單氧化酵素B次單元體與銅離子的高度親合力,我們使用分子生物的技術在大腸桿菌TB1菌株的細胞膜上表現了此蛋白質,並且利用西方式點墨法與蛋白質電泳上,證實了我們的轉殖是成功的;隨後我們將此基因轉殖進入另外一株高銅耐受性的大腸桿菌BCRC®50305,並且在含有高銅離子濃度下的營養液中,培養此株大腸桿菌,並表現微粒體甲烷單氧化酵素B次元體。我們發現在高銅離子濃度下表現此蛋白質時,大腸桿菌的細胞膜會大量增生,這種行為非常類似嗜甲烷菌在培養時銅離子濃度逐漸增高時的行為。並且兩種細菌在此時,也都會在膜上堆積大量還原態的銅離子。這些現象也分別被電子顯微鏡與X光吸收光譜所證實。此外,表現重組微粒體甲烷單氧化酵素B次單元體時,大量增生的膜系統,並未能形成細胞膜的結構,而是以缺乏組織的方式堆積在細胞內部,形成一個類似胞器的新的細胞區間,並且在銅離子缺乏時導致細胞死亡。 此篇論文主要闡述銅離子與重組微粒體甲烷單氧化酵素B次元體間如何交互作用,導致細胞內產生各種不同的生理變化,與重組微粒體甲烷單氧化酵素B次元體,這一蛋白質在不同濃度銅離子下的不同摺疊狀態。 The pmoB sub-domian of pMMO from Methylcoccus capsulatus (Bath) exhibits strong affinity towards reduced copper ions. It was considered that the C-terminal aqueous sub-domain of the protein could accommodate 9-12 reduced copper ions to serve as a reservoir of reducing equivalents for methane activation in the holo pMMO enzyme. However, the recent X-ray crystal structure did not show any features corresponding to these copper ions. One explanation was that these copper ions were lost when the preparation was grown under high oxygen tension or harsh purification condition used. To prove the high copper affinity of this sub-domain, we transferred the subunit B of pMMO into the membrane of E. coli TB1 by exploiting the techniques of molecular biology. The pMMO pmoB was introduced as a fusion protein with Maltose-bind protein. That recombinant process for the over-expression had successfully occurred in the membrane was evidenced by the western blotting with the detection of MBP fusion. The gel electrophoresis analysis also gave rise to the correct molecular weight pattern in the 1D SDS PAGE. Subsequently, we also transformed the designed vector with pmoB insertion into the E. coli strain BCRC®50305. BCRC®50305 belongs to one of the strains originating from the copper tolerant species W3110. The bacteria could be grown in the growth media up to 3.1 mM CuSO4 concentration. We subjected the strains with the pmoB gene to grow in LB buffer containing 1 mM Cu(II) ions. Under high copper ion stress, the cellular membranes of E. coli behaved like the ones in Methylococcus capsulatus (Bath) with abundant membrane accumulation and high content of reduced copper contents. These results were confirmed by electron microscopy and X-ray absorbance spectroscopy, where we discovered that the recombinant pmoB subunit had introduced into a much less structured membrane system within the cell.
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