營養元素磷的攝取主要是以磷酸 (inorganic phosphate, Pi) 的形式經由細胞膜磷酸轉運系統 (phosphate transporter, Pit) 進入細胞中。磷酸轉運膜蛋白利用細胞膜裡外磷酸濃度的梯度產生膜電位差，進行次級主動轉運 (secondary active transporter)，將磷酸和陽離子 (Na+或H+) 離子共同轉運。來自海棲熱袍菌 (Thermotoga maritima) 的磷酸轉運膜蛋白 (phosphate permease) TmPit具有402個胺基酸，其理論分子量計算為44,089 Da。利用生物資訊程式預測TmPit具有10~12個穿膜二級結構 (transmembrane )，其詳細生物功能及分子結構還不清楚，尚待研究。本研究中我們已利用酵母菌異源系統成功大量表現TmPit，順利地用非離子型的介面活性劑DDM (n-dodecyl-β-D-maltoside) 將TmPit 從微粒體 (microsome) 中萃取出來。接著利用各式特性的管柱層析法純化TmPit，並提升 TmPit 純度。在逆向原態膠體電泳 (reverse native PAGE electrophoresis )、粒徑管柱層析法 (size exclusion chromatography, SEC) 及超高速離心分析 (analytical ultracentrifugation, AUC) 實驗偵測TmPit是單一構型的雙聚體 (dimer) 存在於水溶液中。圓偏光二色光譜 (circular dichroism, CD) 實驗結果顯示TmPit其二級結構主要為α-helix。目前利用酵母菌異源系統已可得到適當量 (0.5 mg/8L) 及一定純度的TmPit蛋白質，但是TmPit的純度與產量均需再提升。正藉由懸滴蒸汽擴散法 (vapor diffusion hanging-drop method) 進行TmPit結晶實驗。希望利用生物物理的方法對TmPit的生物功能進行探討，進一步可利用藉由晶體學方法決定TmPit分子的結構。

Nutrient phosphorus is existed as inorganic phosphate (Pi) that can be uptake by phosphate transport (Pit) system into the cell. Phosphate transporter (Pit) be used the membrane potential from the concentration gradient of phosphate for secondary active transport to be a phosphate and cation (Na+ or H+) co-transporter. The residues number and therotical molecular weight of Thermotoga maritima phosphate permease (TmPit) are 402 residues and 44,089Da, respectively. The analysis of bioinformatics showed that TmPit is composed of 10 - 12 transmembrane domains, and the detailed biological function and molecular structure are still not clear. In this study, we have successfully overexpressed the TmPit in the yeast heterologous expression system, and extracted TmPit from microsome by a non-ionic detergent, DDM (n-dodecyl-β-D-maltoside). We used different types of chromatography to improve the purity of TmPit. The analyses of reverse native PAGE electrophoresis, size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) showed that TmPit existed as a dimer with a single conformation in an aqueous solution. Circular dichroism (CD) experiment showed that the secondary structure TmPit mainly constructed by α-helix. Today, TmPit could be expressed and purified in the yeast heterologous systems (0.5mg/8L), but the purity and yield have to be improved. We crystallized TmPit by using vapor diffusion hanging-drop method. In the future, we hoped to use the biophysics and crystallography to study the biological functions and molecular structure of TmPit.

中文摘要 I
Abstract II
第一章 1
前言 1
1.1磷酸 1
1.2磷酸轉運膜蛋白 1
1.3人類磷酸轉運膜蛋白 2
1.4植物磷酸轉運膜蛋白 2
1.5細菌磷酸轉運膜蛋白 3
1.6海棲熱袍菌磷酸轉運膜蛋白 4
第二章 6
材料與方法 6
2.1基因選殖 6
2.2酵母菌製備 6
2.3TmPit轉殖及TmPit誘導 7
2.4 TmPit微粒體 7
2.5 TmPit萃取 8
2.6 TmPit純化 9
2.7 TmPit 結晶 9
2.8 TmPit分析鑑定 10
第三章 14
結果與討論 14
3.1序列比對及穿膜預測分析 14
3.2TmPit表現 15
3.3TmPit萃取及純化 16
3.3TmPit粒徑管柱層析法及超高速離心分析 17
3.5TmPit圓偏光二色光譜 18
3.5 TmPit 結晶 19
第四章 20
結論 20
圖 21
文獻 35

文獻
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