磷酸 (Pi)具有組成分子構造、能量傳遞、磷酸化修飾、維持體液酸鹼平衡等功能，對於生物體是不可或缺的元素。磷酸轉運膜蛋白 (phosphate transporter, PiT)是負責將磷酸轉運進入細胞內的蛋白，在溶質載體家族 (Solute carrier family, SLC)中屬於SLC20家族。磷酸轉運膜蛋白存在於許多物種之中，包括人類、大腸桿菌、酵母菌、阿拉伯芥等。磷酸轉運膜蛋白含有三個結構域，N端PD1131、胞質內環及C端PD1131結構域，其中在N端與C端PD1131結構域中，皆有一段核心共有序列“GANDVANA”，被認為和磷酸結合轉運能力有關，為磷酸轉運膜蛋白家族重要特徵。海棲熱袍菌磷酸轉運膜蛋白 (TmPiT)與原核和真核生物的磷酸轉運膜蛋白具有22-53%及31-41%的序列一致度 (identity)。本研究進行海棲熱袍菌磷酸轉運膜蛋白的分子結構與生物功能之研究。我們利用酵母菌異源表達系統製備TmPiT蛋白及三個突變蛋白(TmPiT-E49A、TmPiT-C248A與TmPiT-H254A)並用加熱法、鎳離子樹脂管柱與粒徑管柱層析法純化，獲得到高純度的TmPiT蛋白及三個突變蛋白。我們利用微量熱泳動實驗 (MST)來測定TmPiT蛋白及三個突變蛋白的磷酸結合能力。微量熱泳動實驗 (MST)結果顯示三個突變蛋白的磷酸結合能力均被降低。我們將純化的TmPiT蛋白與三個突變蛋白製作成蛋白脂質體 (proteoliposome)，並利用32P轉運 (32P uptake)實驗來測定TmPiT蛋白與三個突變蛋白的磷酸轉運能力。32P轉運實驗結果顯示三個突變蛋白的磷酸轉運能力均被降低。這些實驗結果顯示E49、C248及H254為重要胺基酸位點，和TmPiT蛋白的磷酸結合與轉運能力有關。
我們使用懸滴式晶體培養方法進行TmPiT突變蛋白的晶體培養。目前已得到三個突變蛋白晶體，TmPiT-C248A晶體經由x-ray繞射得到3.6 Å解析度，晶體分子空間群為單斜晶系 (monoclinic C2)。TmPiT-E49A及TmPiT-H254A蛋白晶體x-ray繞射結果較差，尚需進一步優化晶體培養條件。

Phosphate (Pi) is involved in many functions such as synthesis of molecular structure, energy transfer, phosphorylation modification, and the maintenance of acid-base balance in body etc. Phosphate is an indispensable element for organisms. Phosphate transporter (PiT) transports phosphate into the cell and belongs to solute carrier family, SLC20. Phosphate transporters are found in many species, such as Homo sapiens, E.coli, Saccharomyces cerevisiae, and Arabidopsis thaliana, etc. Phosphate transporters contain the N-terminal PD1131 domain, intracytoplasmic loop domain and C-terminal PD1131 domain. There is a core consensus sequence“GANDVANA” in both N-terminal and C-terminal PD1131 domains and that is an important feature for phosphate binding and transport capacities in PiT family. Thermotoga maritima phosphate transporter (TmPiT) shares highly sequence identity with prokaryotic PiT (22-53%) and eukaryotic PiT (31-41%). In this study, we study the molecular structure and biological function of the phosphate transporter from Thermotoga maritima (TmPiT). We overexpressed TmPiT and three mutants (TmPiT-E49A, TmPiT-C248A and TmPiT-H254A) by Saccharomyces cerevisiae heterologous expression system and purified by hot solved method, Ni2+-NTA resin and size exclusion chromatography to obtain high-purity TmPiT and mutant proteins. The Pi binding capacities of TmPiT and mutants were determined by microscale thermophoresis (MST) experiment, which results show that the binding capacity of Pi were reduced in three mutant proteins. The Pi transport capacities of TmPiT and mutants were determined by reconstituted proteoliposome 32P uptake experiment, which results show that the transport capacity of Pi were reduced in three mutant proteins either. These experimental results show that E49, C248 and H254 are important residues, which are related to TmPiT Pi binding and transport capacities.
TmPiT mutant proteins crystals were cultivated by vapor diffusion hanging drop method. Up to date, the TmPiT-C248A crystal diffracted to 3.6 Å resolution and belongs to the monoclinic C2 space group. However, TmPiT-E49A and TmPiT-H254A crystals reveal a poor resolution and we are improving the crystallization conditions now.

中文摘要-----------------------------------------I
Abstract----------------------------------------III
誌謝---------------------------------------------V
第一章、簡介--------------------------------------1
1.1 磷酸-----------------------------------------1
1.2 磷酸轉運蛋白----------------------------------1
1.3 溶質載體家族 (Solute carrier family, SLC)-----2
1.3.1 SLC34家族 (第II型磷酸-鈉共轉運蛋白)----------3
1.3.2 SLC20家族 (第III型磷酸-鈉共轉運蛋白)---------3
1.4 人類磷酸轉運膜蛋白 (hPiT)----------------------4
1.5 植物磷酸轉運膜蛋白 (AtPht2)--------------------5
1.6 真菌磷酸轉運膜蛋白 (ScPho89)-------------------5
1.7 原生生物磷酸轉運膜蛋白 (PfPiT)-----------------6
1.8 古細菌磷酸轉運膜蛋白 (MsPiT)-------------------6
1.9 細菌磷酸轉運膜蛋白 (EcPiT)---------------------6
1.10 海棲熱袍菌磷酸轉運膜蛋白 (TmPiT)--------------7
第二章、實驗材料與方法-----------------------------8
2.1 基因選殖與酵母菌勝任細胞製備--------------------8
2.2 酵母菌轉型作用--------------------------------9
2.3 酵母菌表現TmPiT蛋白---------------------------9
2.4 製備酵母菌微粒體------------------------------10
2.5 純化TmPiT蛋白--------------------------------11
2.6 微量熱泳動 (microscale thermophoresis, MST)--12
2.7 製備蛋白脂質體 (proteoliposome)---------------13
2.8 磷酸轉運實驗 (32P uptake assay)---------------14
2.9 晶體培養-------------------------------------14
2.10 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (SDS-PAGE)----15
2.11 西方墨點法 (western blot)--------------------15
2.12 布拉德福蛋白質定量法 (bradford protein assay)-16
第三章、實驗結果----------------------------------17
3.1 TmPiT蛋白序列比對-----------------------------17
3.2 TmPiT表現與純化-------------------------------18
3.3 TmPiT蛋白脂質體製作---------------------------20
3.4 TmPiT生物功能之測定---------------------------21
3.4.1 磷酸結合能力測定----------------------------21
3.4.2 磷酸轉運能力測定----------------------------22
3.5 TmPiT蛋白晶體培養-----------------------------22
3.6 TmPiT突變蛋白 (E49A、C248A與H254A)之蛋白表現---23
3.6.1 TmPiT-E49A突變蛋白-------------------------23
3.6.2 TmPiT-C248A突變蛋白------------------------24
3.6.3 TmPiT-H254A突變蛋白------------------------26
第四章、討論--------------------------------------28
圖表---------------------------------------------31
參考文獻-----------------------------------------45

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