膜蛋白為座落在細胞膜上的蛋白質，進行養分的吸收及儲存、廢物的排放等重要生化功能，是細胞與外界及胞器與胞器之間物質互通及訊息傳遞的重要介質。截至2013年，被解析出的膜蛋白分子結構數目約只佔所有已知的蛋白分子結構(約十萬個)的1 %，這顯示出膜蛋白分子結構的研究仍是結構生物領域的一大挑戰。本論文實驗目的為進行膜蛋白的異源表達、蛋白質純化與製備，希望利用晶體學方法進行分子結構的研究。磷 (P) 對於生物體的成長和發育是一種非常重要的元素，是組成生物膜、核醣核酸等生物分子的必要元素。植物體中所需要的磷是以無機磷酸鹽 (inorganic phosphate) 的方式存在，無機磷酸鹽在植物中如何運輸、調節、以及有效率地利用是一個相當重要的研究課題。本研究將以阿拉伯芥 (Arabisopsis thaliana) 及水稻 (Oryza sativa) 的磷酸運轉膜蛋白 (PHO1) 為研究對象，進行膜蛋白表達與純化系統的建立。文獻指出阿拉伯芥及水稻pho1基因變異下，無機磷酸鹽會堆積在根部，無法運送至莖部及其他部位，導致植株凋亡，故PHO1在磷酸鹽平衡中扮演一個重要角色。PHO1具有一親水性的SPX功能域及一疏水性的EXS功能域，其磷酸運轉的功能被報導是位於EXS功能域。PHO1-EXS分子量約為46 kDa，預測有十個穿膜區域，其詳細的功能及分子結構資訊尚待研究。我們已成功在酵母菌 (Saccharomyces cerevisiae) 蛋白質異源表達系統中克隆、表達出PHO1-EXS並利用介面活性劑將PHO1-EXS自細胞膜溶出，得到可溶性的膜蛋白。接著利用各種層析法進行分離純化PHO1-EXS膜蛋白。由逆向原態膠體電泳 (Reverse Native PAGE) 的實驗指出PHO1在水溶液中具有單一構形。由粒徑管柱層析法 (size exclusion Chromatography, SEC) 的實驗顯示PHO1在水溶液中為雙元體構型。目前我們對PHO1分子的生物物理特性已有一初步的了解，但為了要進行晶體學的實驗，PHO1的產量、純度及穩定度還需要提升。

Phosphate is an essential mineral in both prokaryotic and eukaryotic living cells. Phosphate is the major component of many bio-molecules such as nucleic acids, nucleoside triphosphate and many membrane lipids. Phosphate is also key components in enzyme activity regulation and signal transduction pathways. In plants, phosphate homeostasis is a complicated network, in which phosphate transportation across biological membranes is a major component. Several phosphate transporters are membrane proteins and mainly locate at plasma membrane or endomembrane system that in charge of phosphate acquisition and efflux. Phosphate transporters in plants can be classified into three groups, high affinity phosphate transporter, low affinity phosphate transporter and phosphate exporter. PHO1 is the first protein to be correlated with phosphate export. The detail molecular mechanism of PHO1 is still not clear and there is no three dimensional structure of PHO1. PHO1 contains the N-terminal soluble domain, SPX and the C-terminal transmembrane domain, EXS domain. EXS domain only is reported to have a function of phosphate export. The molecular weight of PHO1-EXS is about 46 kDa. It is predicted that PHO1-EXS might have 10 transmembrane helices. In this study, we successfully cloned, expressed Arabisopsis thaliana and Oryza sativa PHO1-EXS by yeast heterologous protein expression system. Arabisopsis thaliana and Oryza sativa PHO1-EXS was solubilized by detergent and purified by immobilized metal ion affinity chromatography (IMAC). Size-exclusion chromatography (SEC) indicates that purified PHO1-EXS form dimer in solution. Reverse Native PAGE indicate that purified PHO1-EXS is in a single conformation in solution. Also, the pre-crystallization trials are undergoing. However, the purity and quantity of PHO1-EXS still need to be improved in order to do the structural study by crystallography method in the future.

Introduction 2
Material and methods 5
Heterologous expression of AtPHO1-EXS in Escherichia coli 5
1. Cloning of AtPHO1-EXS cDNA to expression vector pET-28a 5
2. Transformation and expression 5
3. Preparation of E. coli inverted membrane vesicle 6
Heterologous expression of AtPHO1-EXS and OsPHO1-EXS in S. cerevisiae 7
1. Cloning of AtPHO1-EXS cDNA and OsPHO1-EXS cDNA into expression vector pYES2 7
2. Transformation and expression 7
3. Preparation of S. cerevisiae microsome 8
Protein quantification 9
SDS-PAGE and Western blotting 9
Membrane protein solublization and purification 10
Size-exclusion chromatography 11
Protein crystallization 11
Results and Discussion 12
Bioinformatics analysis of plant PHO1 12
Heterologous expression of PHO1-EXS in E. coli 14
Heterologous expression of PHO1-EXS in yeast 15
Solubilization of PHO1-EXS 17
IMAC purification of PHO1-EXS 18
Size-exclusion chromatography 20
Crystallization of PHO1-EXS 22
Conclusion 23
References 25
Figures……………………………………………………………………………….30

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