胃幽門螺旋桿菌中的細胞結合因子 (HpCBF2) 屬於一種毒素因子，能夠透過第四型類鐸受體 (Toll-like receptor 4) 活化胃上皮細胞中的上皮生長因子受體 (EGFR) 進一步造成異常的細胞增生。 HpCBF2 蛋白含有兩個功能區，一為肽基脯胺酸順反異構酶功能區 (PPIase domain)，具有催化多肽上的脯胺酸胜肽鍵由順式轉換成反式的功能。另一為伴隨蛋白功能區(chaperone domain)，會參與蛋白質的折疊，影響蛋白質折疊速率的決定步驟，在細胞外膜蛋白成熟機制占有重要的地位。HpCBF2分子量為31,910 Da，我們已成功大量表現及純化，每公升 LB 培養液可得約 25 mg HpCBF2 蛋白，在水溶液中以二聚體聚合存在，二聚體為其功能型。我們已培養得到HpCBF2-apo晶體，以 HKL2000 程式處理並整合繞射數據，其空間群 (space group) 為P3121。晶格參數為a = 60.9 Å, b = 60.9 Å, c = 363.7 Å，晶格中每個不對稱單元中含有兩個 HpCBF2分子，每個單體具有兩個功能區， Chaperone功能區由胺基酸1-147及262-299組成， PPIase功能區則由胺基酸157-253組成。Chaperone功能區中兩個單體會經由 α螺旋互相纏繞而形成功能區調換 (domain swapped) 的結構。我們利用核磁共振光譜實驗 (NMR)決定其脯胺酸順反異構酶的活性，在2D NOESY光譜實驗中計算得到 HpCBF2蛋白的順反異構化反應速率常數 (kex) 為90 s-1，且在加入PPIase 的抑制劑Indole-2-carboxylic acid (I2CA) 明顯降低 HpCBF2蛋白脯胺酸胜肽鍵順反異構化的能力，約為控制組的 25%，證實 I2CA為HpCBF2蛋白順反異構化的抑制劑。這些實驗結果提供了胃幽門螺旋桿菌 HpCBF2蛋白功能的資訊及其三度空間的蛋白分子結構，有助於了解 HpCBF2 蛋白的生物功能與可能的作用機制。

The cell binding factor 2 of Helicobacter pylori (HpCBF2) is a virulence factor and transactivates epidermal growth factor receptor (EGFR) through TLR4 in gastric epithelial cells and lead to abnormal cell proliferation. There are two different functional domains in the HpCBF2, the peptidyl-prolyl cis-trans isomerase (PPIase) domain and chaperone domain. PPIase domain catalyzed cis-trans isomerization of proline peptide bond on oligopeptide. The other domain, chaperone domain also plays an important role in outer membrane protein maturation and may participate in protein folding at the rate-limiting step. HpCBF2 has been overexpressed and purified with the yield is about 25 mg/L LB. The molecular weight of HpCBF2 is 31,910 Da. HpCBF2 forms a dimer in solution and dimer form is the functional type. We have crystallized the HpCBF2-apo crystal successfully. HpCBF2-apo crystal belongs to P3121 space group with cell parameters a = 60.9 Å, b = 60.9 Å, c = 363.7 Å, at 3.6 Å resolution by using HKL 2000 to analyze X-ray diffraction data. There are two molecules per asymmetric unit. Each monomer contains a N-terminal chaperone domain (residues 1-147；262-299) and a C-terminal PPIase domain (residues 157-253). In the HpCBF2-apo dimer, the chaperone domains are interlocked together and form a domain-swapped architecture. The PPIase domain catalyzes transformation of trans and cis isomers of proline peptide bond in oligopeptide. From Nuclear Magnetic Resonance Spectroscopy (NMR), according to 2D NOESY spectrum, we calculate the PPIase activity exchange constant (kex) of HpCBF2 is 90 s-1. Then we perform the inhibition assay by 2D NOESY spectrum using Indole-2-carboxylic acid (I2CA), the inhibitor of HpCBF2, and calculate the crosspeak intensity is a quarter of the control group, obviously reducing the PPIase activity. The results provide a structural detail of the function of HpCBF2 and are helpful to understand the biological
function and mechanism of HpCBF2.

中文摘要………………………………………………………………………… 3
英文摘要………………………………………………………………………… 4
誌謝……………………………………………………………………………… 5
第一章 前言
1-1胃幽門螺旋桿菌 (Helicobacter pylori)………………………………… 6
1-2 脯胺酸順反異構化反應 (Prolyl cis-trans Isomerization)……………. 6
1-3 肽基脯胺酸順反異構酶 (PPIase)……………………………………… 7
1-4 Parvulin 家族之 PPIase 相關蛋白質…………………………………. 9
1-5肽基脯胺酸順反異構酶的抑制劑及受質……………………………..... 10
1-6胃幽門螺旋桿菌細胞結合因子 (HpCBF2)……………………………. 10
第二章 材料與方法
2-1 基因選殖………………………………………………………………... 12
2-2 HpCBF2 蛋白表現與純化
2-2-1 HpCBF2 蛋白表現…………………………………………………... 12
2-2-2 HpCBF2 蛋白純化…………………………………………………... 13
2-3 超高速離心分析………………………………………………….……. 13
2-4 脯胺酸順反異構酶活性分析……………………………………..…… 14
2-5 蛋白晶體培養……………………………………………………..…… 14
2-6 X-光繞射數據的收集與分析…………………………………….…….. 15
第三章 實驗結果
3-1 序列比對……………………………………………………………….. 16
3-2 蛋白質純化與生物特性……………………………………………….. 16
3-3 脯胺酸順反異構酶 (PPIase) 活性分析…………………………….... 17
3-4 PPIase活性抑制分析………………………………………………….. 19
3-5 蛋白質晶體的形成…………………………………………………….. 19
3-6 HpCBF2-apo 蛋白晶體結構………………………………………….. 20
第四章 討論
4-1 蛋白質純化與生物特性………………………………………..……… 21
4-2 PPIase 活性分析………………………………………………………. 21
4-3 HpCBF2-apo 與HpCBF2-I2CA complex 晶體結構比較………... 22
圖………………………………………………………………………………. 24
表………………………………………………………………………………. 40
第五章 參考文獻……………………………………………………………... 44

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