液泡質子傳送焦磷酸水解□ (簡稱 V-PPase; EC 3.6.1.1) 是液泡膜上的質子傳送系統之一，它能夠利用水解焦磷酸時所產生的能量將質子由細胞質傳送到液泡中，產生液泡膜內外質子梯度及膜電位差異，藉以驅動其他次級主動運輸的進行。V-PPase是分子量約80 kD的單一胜□鏈所構成的同雙次體，並且擁有大約16個穿膜區段。V-PPase的詳細結構及酵素作用機制目前仍尚未釐清，因此利用蛋白質晶體學等實驗方式得到高解析度的蛋白質結構資料，將會是相當重要的突破。在本研究中，我們利用酵母菌異體表達系統大量生產綠豆 (Vigna radiata) V-PPase，並且根據沉降係數的差異，分離出富含V-PPase的酵母菌膜質部分（Membrane fraction）。我們成功利用介面活性劑DDM (全名為n-dodecyl-β-D-maltoside) 將穿膜蛋白質由膜質中萃取為水溶性蛋白質，接著結合金屬螯合親合性層析法 (Metal chelate affinity chromatography) 以及體積排除層析法 (Size-exclusion chromatography) 純化得到水溶性的V-PPase 蛋白質。此純化後蛋白質仍具有水解焦磷酸之酵素活性。根據實驗結果，我們相信藉由此表達及純化系統可得到高純度及具有活性構型的V-PPase蛋白質。目前我們已開始利用蒸氣擴散法進行蛋白質結晶實驗，期待能成功利用蛋白質晶體學的方法解出高解析度之V-PPase蛋白質結構。

Vacuolar H+-pyrophosphatase (abbreviated V-PPase; EC 3.6.1.1), one of the proton translocation system in the vacuolar membrane, hydrolyzes pyrophosphate and uses the energy for translocating the protons from cytosol into vacuole. The proton translocation causes the pH gradient and membrane potential difference across the vacuole membrane for driving the secondary active transport in the vacuole membrane. V-PPase is a homodimer composed of a single polypeptide with molecular mass of about 80 kD, and there are approximately 16 transmembrane domains in each monomer. The precise protein structure and enzymatic mechanism are still unknown; it would be a quite important breakthrough to solve the high resolution V-PPase structure by X-ray crystallography. In this study, we have overexpressed the mung bean (Vigna radiata) V-PPase in the yeast heterologous expression system and isolated the V-PPase enriched membrane fraction according to the difference of sedimentation coefficient. We have successfully extracted the membrane proteins from membrane fraction by using DDM (n-dodecyl-β-D-maltoside) as the detergent and utilized metal chelate affinity chromatography and size-exclusion chromatography to purify the solubilized V-PPase. Upon purification, the V-PPase still maintains the enzymatic activity of pyrophosphate hydrolysis. Accordingly, we are convinced that these expression and purification protocols were feasible for producing high purity V-PPase proteins with functional conformation. We are currently developing crystallization screening by using vapor diffusion method and the progress for X-ray protein crystallography is promising at this stage.

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