磷在生物體中常以磷酸的形式存在，其在生物體中扮演許多重要的角色，
磷酸轉運穿膜蛋白(PiT)能將磷酸運至細胞膜內以維持胞內磷酸的恆定。在溶質運送蛋白(Solute carrier, SLC)家族中， SLC20是一群將磷與鈉協同運輸的磷酸轉運蛋白，其有著高度保留的內部重複序列(GANDVANA/S)且廣泛存在於生物界的細胞中。然而現在仍然缺乏SLC20家族或其同源蛋白的分子結構來解釋SLC20在磷酸轉運和一些疾病中的機制。海棲熱袍菌(Thermotoga maritima)的磷酸轉運膜蛋白(TmPiT)具有高度保留的內部重複序列，且與原核和真核生物的磷酸轉運膜蛋白都有高度的相似性。它在本研究中被用來作為探討磷酸轉運膜蛋白分子結構和功能。我們成功的分離出在酵母菌(Saccharomyces cerevisiae)上異種表達的TmPiT蛋白。並藉由加入其受質磷酸和調整熱解法中的溫度去改善TmPiT的純度，這麼做能穩定TmPiT並降低多聚體的形成。為了得到更高品質的晶體，不同的介面活性劑 (detergent) 被應用在TmPiT的溶解和結晶中，從結果上來看，TmPiT被鑲嵌在較大膠束大小(micelle size) 的介面活性劑上能獲得更高解析度 (3.7 Å) 但在空間晶格中較低對稱性 (C2 space group) 的晶體。然而就目前而言晶體仍需改進以獲得更好的X-ray繞射資料，並需進一步的去解出其相位問題。

Living cells require inorganic phosphate (Pi) as a critical material for many biology functions. Inorganic phosphate transporters (PiT) are a kind of membrane protein that can transport Pi across the membrane to maintain phosphate homeostasis. In the all of solute carrier (SLC) family, PiT belongs to the SLC20 that are Na+-coupled Pi cotransporter. It has a conserved internal repeat sequence, GANDVANA/S, and exists in all kingdom cells. However, it still lacks molecular structure of SLC20 or their prokaryotic homologs to explain its certain function role in Pi transport and some diseases. The PiT from Thermotoga maritima (TmPiT) also has conserved internal repeat sequence and shares high identity with prokaryotic and eukaryotic PiT. It is utilized to study structure and function of PiT in this research. We have successfully isolated TmPiT protein which was heterologously expressed in Saccharomyces cerevisiae. The purity of TmPiT was improved by adding its substrate (Pi) and adjust the heating temperature in the Hot-Solve method which can stabilize TmPiT and decrease the oligomer formation. To get a higher quality crystal, different detergents were applied in solubilization and crystallization of TmPiT. The results showed TmPiT embedded in larger micelle size detergent could form the higher resolution (3.7Å) but lower symmetry (C2 space group) crystal. But for now, the quality of X-ray diffraction dataset still needs to be improved, and the phase problem need to be determined.

Contents
Abstract I
中文摘要 II
誌謝 III
Chapter 1 Introduction 1
1.1 Phosphate 1
1.2 Phosphate transporter 2
1.2.1 SLC34 family (type II Na+ -coupled Pi transporters) 3
1.2.2 SLC20 family (type III Na+ -coupled Pi transporters) 4
1.2.3 PiT family 5
1.3 Phosphate transporter in mammal 5
1.4 Phosphate transporter in plant and fungi 6
1.5 Phosphate transporter in protist and archaea 8
1.6 Phosphate transporter in bacteria 8
1.7 Phosphate transporter in Thermotoga maritima 9
Chapter 2 Material and Methods 11
2.1 TmPiT transformation 11
2.2 TmPiT expression 12
2.2 Isolation of S. cerevisiae microsome 12
2.3 Microsome concentration quantification 13
2.4 .1 Protein isolation with Hot-Solve method 14
2.4.2 Protein purification with nickel column 14
2.4.3 Protein purification with size-exclusion column 15
2.5 SDS-PAGE 15
2.6 Western blotting analysis 16
Chapter 3 Result 18
3.1 Sequence analysis and topology of TmPiT 18
3.2 TmPiT expression 20
3.2.1 TmPiT isolation 21
3.2.2 TmPiT purification improvement 22
3.3 Different detergent application in TmPiT 24
3.3.1 DDM embedded the TmPiT 25
3.3.2 DM embedded the TmPiT 26
3.3.3 DDMB embedded the TmPiT 26
3.3.4 Cymal-7, OG and NM embedded the TmPiT 27
3.4 TmPiT crystallization in different detergent 28
3.4.1 TmPiT crystallization in DDM 28
3.4.2 TmPiT crystallization in DM 29
3.4.3 TmPiT crystallization in DDMB 30
3.4.4 TmPiT crystallization in Cymal-7 31
Chapter 4 Conclusion 32
Figure and Table 33

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