人類海藻糖水解酶重組蛋白之表達

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2014

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海藻糖 (trehalose) 是一種非還原性的雙糖,由兩個葡萄糖分子以 α,α-1,1- 糖苷鏈結形成,存在於多種生物體中,除了可以做為生物能量來源,亦有穩定蛋白質與細胞膜等重要生理功能。海藻糖水解酶 α,α-Trehalase (TreH) (EC 3.2.1.28),可將海藻糖水解成兩個葡萄糖,廣泛的存在於微生物與動植物中。人體不同組織中亦含有 TreH,例如小腸與腎臟絨毛膜的 TreH 負責海藻糖的水解以利吸收,另外,血漿中亦含有TreH,有研究顯示血漿中具有高 TreH 活性的人較容易罹患糖尿病。然而,目前為止對於人類 TreH 的生化特性、功能與結構的研究非常稀少。本研究將選殖的兩種人類海藻糖水解酶 isoform1 (hTreH1) 和 isoform2 (hTreH2) 的基因,利用三種蛋白表達系統,包括原核生物的大腸桿菌 (Escherichia coli) 表達系統、真核生物的畢赤酵母菌 (Pichia pastoris) 表達系統和昆蟲桿狀病毒表達系統 (baculovirus expressing vector system),以便生產具功能性的重組酵素。其中,兩種 hTreHs 在大腸桿菌表達都形成不可溶的內涵體,以隨機突變技術篩選 80 個突變株,並未獲得可表達水溶性酵素之突變株;在畢赤酵母菌表達出的胞外重組酵素量極微,在培養液中無法偵測到 TreH 活性;而在昆蟲桿狀病毒表達系統中有表達出重組酵素,但是仍然形成不可溶的蛋白沉澱。部分可溶的重組蛋白可以非變性的蛋白萃取條件萃出,然而這些萃出的重組蛋白還是不具活性。本研究所採用的方法中沒有表達出具有活性的重組蛋白,為了得到可溶且具有活性的重組蛋白,需要再進一步做更深入的研究。
Trehalose is a non-reducing disaccharide formed by two glucose molecules linked by a 1α-1α glycosidic bond. It occurs in a wide range of bacteria, fungi, insects, invertebrates and plants. Apart from being as energy source, trehalose has many important physiological functions such as stabilizing proteins and cell membranes. α,α-Trehalase (EC 3.2.1.28) (TreH), which hydrolyzes α,α-trehalose to two glucose molecules, is widespread in nature and found in various human tissues as well as in plasma. It has been reported that people with high plasma trehalase activity are more prone to develop diabetes mellitus than people with low enzyme activity. However, the biochemical property and structure of human TreH have not been determined. In order to produce functional recombinant human trehalases (hTreH), hTreH1 and hTreH2 were expressed in various protein expression systems including prokaryotic E. coli, eukaryotic yeast Pichia pastoris and insect cell systems. In the E. coli expression systems, hTreHs were expressed as inclusion body. Random mutagenesis and screening for improving recombinant protein solubility were performed and no improved mutants were screened among eighty transformants. In Pichia pastoris, the expression level of secretory hTreHs were very low and no activity were detected in the culture medium. In insect cell systems, hTreHs can be expressed, but appeared as insoluble form. Some soluble recombinant hTreHs were extracted under non-denaturing conditions, however in biologically inactive forms. Further investigations are needed to obtain the soluble biologically active forms of recombinant hTreHs.

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海藻糖, 人類海藻糖水解酶, 大腸桿菌, 畢赤酵母菌, 桿狀病毒, sf9 昆蟲細胞, trehalose, human trehalase, E. coli, Pichia pastoris, baculovirus, sf9 cell

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