磷酸轉運蛋白扮演維持胞內磷酸平衡的重要角色以維持細胞功能，例如參與蛋白質的磷酸化修飾以及血液中的緩衝溶液。Solute carrier family (SLC) 共有52個族群，其中的SLC20家族為一群將鈉與磷協同運輸的磷酸轉運蛋白，具有兩種異構型SLC20A1/ PiT1和SLC20A2/PiT2，並被報導指出它們都具有一個共同序列GANDVANA。為了簡化及聚焦在磷酸轉運功能的研究，我們挑選一種叫做海棲熱袍菌的細菌中與HsPiT有同源關係的磷酸轉運蛋白(TmPiT: Thermotoga maritima phosphate transporter)作為研究主題。我們已經成功的利用酵母菌異源表現系統大量表現TmPiT，並以加熱法(Hot solve method)來溶出蛋白、鎳離子親和性管柱(nickel affinity column)、粒徑管柱層析法(size exclusion chromatography)來獲得高純度的蛋白並進行結構生物學的研究。為了增加蛋白的產量及增進膜蛋白TmPiT的純度，我們更換用於酵母菌異源表現的培養基，並使用改良的加熱法(modified hot solve method) 去除雜蛋白。然而，在粒徑管柱層析法(size exclusion chromatography)的實驗中我們發現TmPiT在detergent n-Dodecyl-β-D-Maltoside (DDM)或7-Cyclohexyl-1-Heptyl-β-D-Maltoside (Cymal-7)的包覆下具有兩種形式。最近我們以蒸汽擴散法(vapor diffusion)得到一個適合TmPiT長晶的條件 然而Ｘ-光繞射的解析度並不夠用於建構蛋白結構。我們亦嘗試另一種界面活性劑n-Decyl-β-D-Maltoside來包覆TmPiT並用於結晶學研究。

Phosphate transporter plays an important role to maintain intracellular phosphate homeostasis for cell functions, such as protein phosphorylation and buffer solution in blood. The solute carrier (SLC) families contain 52 groups and PiTs belong to SLC20 are Na+-coupled Pi cotransporters which have two isoforms, PiT1/SLC20A1 and PiT2/SLC20A2, and they have been reported to have a consensus sequence GANDVANA. To simplify and focus on phosphate transport function, we selected a protein called TmPiT, a homologous of HsPiT from a bacteria called Thermotoga maritima, to study. We have successfully overexpressed TmPiT by yeast heterologous expression system, and the hot solve method during solubilization, nickel affinity column and size exclusion chromatography (SEC) are utilized to obtain pure protein to study structural biology. To enlarge the yield and improve the purity of TmPiT membrane protein, we change the yeast cultural medium and exploit a modified hot solve method to remove non-target protein. However, TmPiT exists two forms from the SEC via detergent n-Dodecyl-β-D-Maltoside (DDM) and 7-Cyclohexyl-1-Heptyl-β-D-Maltoside (Cymal-7). Recently, we have obtained a condition that is suitable for TmPiT crystallization by vapor diffusion. The native crystal dataset was collected; however, the quality of the dataset is not good for structural determination. The other detergent n-Decyl-β-D-Maltoside (DM) is applied to improve the TmPiT for crystallization.

Contents
1. Abstract: 1
2. 中文摘要: 2
3. 誌謝 3
4. Introduction: 4
Phosphorus 4
Phosphate transporter 4
Phosphate transporter in Human 5
Eukaryotic phosphate transporter, PiPT 7
Phosphate transporter in archaea 8
Phosphate transporter in prokaryote 8
Phosphate transporter in Thermotoga maritima 8
5. Material & Method: 10
Yeast transformation and Protein expression 10
Preparation of S. cerevisiae microsome 11
Microsome quantification 11
Hot solve solubilization and purification with nickel column 12
Protein purification with size-exclusion column 13
SDS-PAGE 14
Protein crystallization 14
Analytical ultracentrifugation (AUC) analysis 15
6. Results: 16
Sequence alignment and topology of TmPiT 16
Purification of TmPiT by hot solve and IMAC 17
SDS-PAGE analysis 18
Size exclusion chromatography of TmPiT 19
Dimer and oligomer forms of TmPiT 19
Different condition test for TmPiT 21
TmPiT in DDM for crystallization 22
Different detergent embedded TmPiT 24
TmPiT in Cymal-7 for crystallization 25
TmPiT in DM 26
Analytical ultracentrifugation (AUC) analysis of TmPiT 27
7. Conclusion 29
8. Figures& Table: 30
(n-Decyl-β-D-Maltoside) 32
9. Reference: 52

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