臺灣博碩士論文加值系統

Cyclic di-GMP是一個獨特且廣泛存在於細菌中的二級訊息傳遞分子，細菌藉由這一個訊息傳遞分子控制著複雜的細胞活動。以前對於c-di-GMP受體蛋白的研究主要關注在具有GGDEF、EAL、HD-GYP、PilZ等功能域的蛋白質，但cyclic di-GMP可能的功能尚未完全明瞭。最近，Robert P. Ryan等人利用pull-down assay的方式發現十字花科黑腐病菌 ( Xanthomonas campestris pv. campestris , Xcc ) 中的XcYajQ ( XC_3703 ) 為一重要蛋白，不但會和cyclic di-GMP結合，並可以和其他蛋白產生交互作用，改變這些蛋白的功能。本篇論文主要針對XcOPPs與XcYajQ蛋白複合體的結構與功能分析。XcOPPs是一個Octaprenyl pyrophosphate 合成酶，會將一個法尼焦磷酸( farnesyl pyrophosphate )和五個異戊烯焦磷酸( isopentenyl pyrophosphate )結合，形成一個具有40個碳的長鏈產物，以供泛醌( ubiquinone )和甲基萘醌( menaquinone )的側鏈合成。由於泛醌和甲基萘醌在細菌體內的電子傳遞鏈中具有重要的功能，因此cyclic di-GMP可能藉由影響XcOPPs的酵素活性調節細菌的電子傳鏈速率，改變細菌的生理現象。
本論文利用Biacore方式測定出XcYajQ和XcOPPs之間有強的親和力，達到2.76µM。接下來，再利用小角度散射，分別觀察XcYajQ和XcOPPs的大致外型。又使用EnzChek pyrophosphate assay kit ( Molecular Probes Inc. ) 測量XcOPPs的酵素活性，以及XcYajQ和cyclic-di-GMP的結合如何影響XcOPPs的合成能力。發現當XcYajQ和XcOPPs作用時，會使得XcOPPs的活性提升。而加入cyclic-di-GMP時能讓XcOPPs的活性更加增強。本論文首次證實藉由與XcYajQ的結合，cyclic di-GMP可間接影響XcOPPs的酵素活性。

Cyclic di-guanylate (cyclic di-GMP) is an unique and important second messenger in bacteria which regulates many critical processes include motility, biofilm formation and virulence. But it may play additional functions that are to be uncovered . Recent study found that XcYajQ, a YajQ family protein from the plant pathogen Xanthomonas campestris pv. Campestris (Xcc), is a novel receptor of cyclic di-GMP. Importantly, XcYajQ is able to interact with a variety of other proteins to alter their function. Octaprenyl pyrophosphate synthase (OPPs) is one of these identified proteins which can interact with XcYajQ with strong affinity. XcOPPs catalyzes the formation of a C40 long-chain product OPP that serves as the side chain of ubiquinone and menaquinone by using one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules. It is likely that interaction of XcYajQ with XcOPPs may influence the XcOPPs activity.
We have expressed and purified XcYajQ and XcOPPs to a hige purity, and used Biacore to measure the affinity between XcYajQ and its two ligands cyclic di-GMP and XcOPPs. XcYajQ is found to exhibit strong affinity toward cyclic di-GMP and XcOPPs with a KD of 0.35µM and 2.76µM, respectively. They are thus good target for structure studies using X-ray crystallography. We also find out that XcYajQ can increase XcOPPs enzyme activity, furthermore, cyclic di-GMP enhance XcOPPs enzyme activity to a higher level by binding to XcYajQ. This dissertation first proves that cyclic di-GMP can indirectly alter XcOPPs enzyme activity by bindind with XcYajQ.  

壹、 前言 1
一、 細菌的二級訊息傳遞分子 cyclic di-GMP 1
二、 Xanthomonas campestris pv.campestris 2
三、 新型cyclic di-GMP受體蛋白XcYajQ 3
四、 XcYajQ的交互作用蛋白XcOPPs 4
貳、 材料與方法 6
一、 構築目標蛋白質及載體 6
(一)、 設計目標引子 6
(二)、 萃取染色體DNA 7
(三)、 目標基因聚合酶連鎖反應 7
(四)、 以膠體電泳檢查目標蛋白基因片段 8
(五)、 純化基因片段 9
(六)、 置備N端double His6-tag insert 及載體 9
(七)、 置備C端His6-tag insert 及載體 11
(八)、 勝任細胞之製備 13
(九)、 轉殖作用(Transformation) 13
(十)、 目標基因之定序 13
二、 蛋白質之大量表現及純化 15
(一)、 目標蛋白的大量表現 15
(二)、 以均質細胞破碎機進行破菌 15
(三)、 Ni-CAM親和性管柱純化目標蛋白質 15
(四)、 用Tobacco Etch Virus ( TEV ) 去除His6-tag 16
(五)、 使用凝膠過濾法純化目標蛋白 16
(六)、 Selenium標定蛋白質之製備 16
(七)、 蛋白濃度的測定 18
三、 XcYajQ和XcOPPs的生物物理測量 19
(一)、 Thermofluor screen 19
(二)、 XcOPPs和XcYajQ複合體的凝膠過濾管柱分析 19
(三)、 生物分子交互作用分析技術 ( Biacore ) 20
四、 結晶條件的篩選 22
(一)、 結晶條件的篩選 22
(二)、 蛋白質與cyclic di-GMP的共結晶實驗 ( co-crystallzation ) 22
(三)、 種晶實驗 ( microseeding ) 23
五、 Small angle X-ray scattering ( SAXS ) 25
(一)、 X光小角度散射實驗 25
(二)、 基本原理 25
(三)、 資料的收集與運算 25
六、 XcOPPs的酵素活性測量 27
(一)、 EnzChek pyrophosphate assay kit 27
參、 結果與討論 29
一、 目標蛋白的選擇 29
二、 目標蛋白的表現 29
(一)、 XcYajQ 29
1. 表現載體之構築、蛋白質的表現與純化 29
2. 蛋白結晶條件的篩選 29
3. XcYajQ的X光小角度散射的結果 30
(二)、 XcOPPs 30
1. 表現載體之構築、蛋白質的表現與純化 30
2. 以Thermofluor screen篩選緩衝液 31
3. 蛋白結晶條件的篩選 31
4. XcOPPs和XcYajQ複合體的凝膠過濾管柱分析 32
5. XcOPPs和XcYajQ的親和力測試 32
6. XcOPPs 的X光小角度散射結果 32
7. XcOPPs的酵素活性 33
8. XcOPPs和XcYajQ的共結晶實驗 33
肆、 結論 35
伍、 參考文獻 36
陸、 附表 41
柒、 附圖 45

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