臺灣博碩士論文加值系統

在基因體計畫的初步完成中，宣告著蛋白質體主宰生命科學研究的時代即將來到。這激勵著我們研發新的技術去從事蛋白質體的研究。本篇論文中，我們結合了先進的奈米科技與傳統利用抑制劑來找尋目標蛋白質的策略，發展出新一代能夠同時標示與純化蛋白質於複雜的生物系統中的分析平台，並希望這項新的應用能夠展現它在蛋白質體學方面的潛力。

苯磺酸胺本是一個碳酸酐酶二的抑制劑，我們以一個離胺酸為骨幹，分別在上面連接上苯磺酸胺與一個環氧基與長碳鏈硫醇而設計出我們目標化合物1。環氧基的目的在於利用它對親核基的活性來加強苯磺酸胺對蛋白質的標示作用，長碳鏈硫醇的目的在於使這個化合物能夠具有組裝於金表面的能力。

化合物1在合成出來後我們將其組裝在32奈米大小的金奈米粒子表面上作為配位基，建構出我們的1號金奈米粒子(GNP-1)，利用元素分析顯示在每一顆GNP-1中我們大約組裝了4000個配位基也就是化合物1於其表面。我們選擇以牛碳酸酐酶II（BCA(II)）作為目標蛋白質，將GNP-1與BCA(II)混合於溶液當中進行辨識與共價標示作用。隨後便利用離心的方式將粒子分離後進行SDS-PAGE的分析與Commasie Blue的染色來觀察粒子是否有攜帶著BCA（II）

在對濃度與時間的探討實驗中，我們發現GNP-1對BCA（II）的標示與偵測作用，最低的偵測極限濃度在3.3~33 nM左右，而標示作用完全進行需要約十分鐘的時間。在蛋白質活性探討的實驗，我們先將BCA(II)加熱使其變性，結果顯示GNP-1對於這個變性的BCA(II)產生更強的標示作用。在選擇性的探討實驗中，GNP-1對其他四種不同的蛋白質並沒有明顯的作用，顯示了GNP-1的專一性。

為了評估GNP-1在蛋白質體的活性，我們將其與BCA(II)以及其他三種蛋白質混合的蛋白質混合物作用。結果顯示GNP-1的確能在混合物中標示BCA(II)。透過離心的方式可GNP-1/BCA(II)與未反應之蛋白質分離純化出來。我們利用MALDI-TOF直接分析GNP-1 /BCA(II)進行蛋白質身份確認。

我們亦合成了化合物2、3作為控制分子並將這些分子修飾在奈米粒子上，希望透過這些分子進而瞭解GNP-1對蛋白質作用的機制。從控制實驗的結果顯示GNP-1對蛋白質的選擇性來自於苯磺酸胺的辨識作用，以及環氧基對蛋白質標示親和力的加強。

The completion of Human Genome Project heralded the beginning of the proteome era. We are urged to develop new technology which shows the potential toward proteomics. By introducing the frontier nano- technology, traditional inhibitor design of targeting enzyme has provided a novel platform for facilitating labeling and purification of protein with high biocomplexity.

Benzene Sulfonamide, a general type of carbonic anhydrase inhibitor combined with both a reactive epoxy group for affinity enhancement on labeling proteins and a thiol tail for anchoring on the gold surface has been synthesized within the scaffold of the tri-functionalized lysine
The functionalized gold nanoparticles GNP-1 was fabricated after grafting compound 1 as ligand on the gold nanoparticles with an average radius of 32nm. Elemental analysis showed that there were about 4000 molecules per particle. Bovine Carbonic Anhydrase II BCA(II) was chosen as a model enzyme. Recognition and affinity binding therefore took place in this micro heterogeneous system containing both GNP-1 and protein solution. GNP-1 was separated from unlabeled protein by centrifugation and analyzed by standard SDS-PAGE and Commasie Blue staining.

Model experiments were carried out at the BCA(II) presence only. In the time- & concentration-dependence experiments, the results showed a 3.3~33 nM range for detection limit for this labeling of BCA(II) and 10 minutes for the labeling to be completed. In the activity-based experiment we denatured the BCA(II) by a pretreatment of heating on 95℃for half an hour. For this denatured BCA(II), GNP-1 displayed higher labeling yield than native one. Selectivity experiment showed no obvious affinity toward four other proteins (BSA, BS-II, RNaseA, and WGA) and proves the recognition occurrence between the designed GNP-1 and BCA(II).

Proteome activity was evaluated by introducing GNP-1 into simple proteins mixture containing BCA(II) and three other proteins. GNP-1 successfully recognized BCA(II) in the mixture and separate them from proteins mixture by centrifugation. And this result was further confirmed by MALTI-TOF for protein identification.

Moreover, ligand structure has been systematically modified as controls for mechanistic investigation during this selective labeling. The result confirms that the selectivity originates from benzene sulfonamide binding to the active site and the epoxide functionality to covalently label the targeted protein.

第一章 緒論…………………………………………………………1
1.1蛋白質體學的介紹 …………………………………………1
1.1-1後基因體時代的來臨…………………………………1
1.1-2 蛋白質體學的起步與發展……………………………2
1.1-3 表現蛋白質體學：二維膠體電泳與質譜儀…………4
1.1-4 功能蛋白質體學………………………………………8
1.1-5 我們發展蛋白質學的願景 …………………………12
1.2 金奈米粒子的介紹…………………………………………13
1.2-1歷史背景………………………………………………13
1.2-2金奈米粒子的合成 …………………………………14
1.2-3自我組裝………………………………………………17
1.2-4物理化學角度來看金奈米粒子溶液…………………19
1.2-5 表面電漿帶(SPB)……………………………………21
1.2-6 金奈米粒子基礎之生物探針………………………23
1.2-7 金奈米粒子在探針發展的優點討論………………26
1.3 蛋白質探針與純化方式之介紹 ………………………………27
1.3-1蛋白質的辨識……………………………………… 27
1.3-2親和力加強的策略……………………………………30
1.3-3 蛋白質辨識作用的運用：以親和力分離來純化蛋白質
…………………………………………………………………33
1.4 以碳酸酐酶為作用目標之金奈米粒子的架構………………36
1.4-1碳酸酐酶之介紹………………………………………36
1.4-2苯磺酸胺抑制劑的發展………………………………37
1.4-3苯磺酸胺衍生物Gong Chen-9的設計與應用……41

第二章 金奈米粒子的設計以及配位基的合成策略探討………44
2.1 GNP-1的設計理論…………………………………………44
2.2 控制實驗與GNP-2及GNP-3的設計理論………………46
2.3 化合物1的拆解以及合成策略介紹………………………48
2.4 化合物4之設計原理與合成………………………………56
2.4-1 正交保護的設計需求………………………………56
2.4-2化合物4之合成方法 ………………………………57
2.5縮酸的活化（化合物7、8）………………………………60
2.6選擇性去保護與耦合 Part I………………………………62
2.7 化合物13的NMR氫譜討論……………………………69
2.8 選擇性去保護與耦合 Part II……………………………73
2.9甘胺酸衍生物化合物3的合成……………………………76

第三章 實驗結果與討論………………………………………80
3.1 金奈米粒子對配位基的裝載能力的討論…………………80
3.2 金奈米粒子的大小與尺寸分佈……………………………86
3.3 蛋白質對金奈米粒子穩定度的影響………………………87
3.4 牛碳酸酐酶II的結構討論…………………………………89
3.5 GNP-1對BCA(II)的活性測試……………………………90
3.6 加熱實驗的延伸……………………………………………97
3.7 控制實驗的結果與討論…………………………………101
3.8蛋白質混合物的純化與MALDI-TOF的實驗結果…………107

第四章 結論…………………………………………………………115
實驗部分………………………………………………………………117
參考文獻………………………………………………………………146
附錄……………………………………………………………………155

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