摘要
蛋白質開關已廣泛應用於以細胞基底的感測器與即時檢測中，其可用於快速分析各類蛋白質、代謝物、核酸，以及酵素活性。目前，蛋白質開關主要依賴兩種開關機制以將目標物結合行為轉化為定量的訊號，即改變螢光分子的光學特性與酵素活性的活化。
在本論文中，我們闡述了一種新的開關機制，其中目標小分子與蛋白質結合後會活化鏈黴親和素與生物素之間的作用以產生訊號。在缺乏目標物的狀況下，生物素化的蛋白質開關呈現關閉構象，而生物素鄰近於蛋白質，施加於其上的龐大空間立障會阻礙生物速與鏈黴親和素的結合。而在開啟的構象時，空間立障遭到移除，使得裸露的生物素可與鏈黴親和素結合。憑藉此種偵測概念，可用不同的染料與檢測技術以偵測天然和基因工程細胞膜表面上的磺胺類小分子藥物。
我們相信此種控制生物素與鏈黴親和素結合的可調控親和力之半合成蛋白質開關設計可成為快速、具特異性、並且靈敏的小分子檢測工具。
 

Abstract
Protein switches have shown wide applications in cell-based protein sensors and point-of-care diagnosis for the rapid analysis of a wide variety of proteins, metabolites, nucleic acids and enzymatic activities. Currently, protein switches rely mainly on two type of switching mechanisms to transduce the target binding event to a quantitative signal, namely changing the optical properties of fluorescent molecules and activation of enzymatic activities.
In this paper, we introduce a new type of switching mechanism in which binding of a small molecule target with the protein activates streptavidin-biotin interaction to generate readout signal. In the absence of a target, the biotinylated protein switch forms a closed conformation where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to prevent the effective binding with streptavidin. In the open form, this steric hindrance is removed, thereby exposing the biotin for streptavidin binding. With this sensing concept, various sulfonamides drugs can be selectively detected on the cell surface of native and genetically engineered cells by using different fluorescent dyes and detection techniques.
We believe that our affinity-tunable semisynthetic protein switch design to regulate streptavidin-biotin binding affinity can be a versatile tool for rapid, specific and sensitive detection of many other molecular targets.

目錄
摘要---------------------------I
Abstract----------------------II
謝誌--------------------------III
目錄---------------------------V
第一章: 緒論--------------------1
1-1. 小分子的介紹---------------1
1-1.1 生物體中的小分子----------1
1-1.2 商業用動物用藥------------2
1-1.3 人類用藥------------------4
1-1.4 磺胺類藥物----------------5
1-2. 自然界偵測小分子之方法------7
1-2.1 G蛋白偶聯受體-------------8
1-2.2 乳糖操縱子----------------9
1-3.人類開發之偵測小分子方法-----10
1-3.1 高性能液相層析儀-----------11
1-3.2 酵素結合免疫吸附分析法------12
第二章: 文獻回顧-----------------14
2-1蛋白質開關 (Protein switch)----14
2-1.1 酵素基礎的蛋白質開關 (Enzyme based protein switch)--------15
2-1.2 螢光共振能量轉移 (Förster resonance energy transfer, FRET)-----18
2-1.3 螢光基礎的蛋白質開關 (Fluorescent based protein switch)--------18
2-2 半合成蛋白-----------------------21
2-2.1 半合成蛋白基底的探針------------21
第三章: 探針設計---------------------23
3-1 探針工作原理設計與結構------------23
3-2 探針各官能基團介紹----------------24
3-2.1 配體端 (Ligand site)-----------25
3-2.2 反應端 (Reaction site)----------26
3-2.3 生物素端 (Biotin site)----------27
3-2.4 探針結構------------------------31
第四章: 實驗結果與討論-----------------33
4-1 探針A、探針B與探針C之合成-----------33
4-1.1 探針A之合成---------------------33
4-1.2 探針B之合成---------------------34
4-1.3 探針C之合成---------------------34
4-2 探針A之實驗結果--------------------35
4-2.1 磺胺類小分子藥物測試-------------37
4-2.2 非磺胺類小分子藥物測試------------35
4-2.3 共培養時間與螢光增益-------------38
4-2.4 螢光基團更換--------------------39
4-2 探針B之實驗結果--------------------40
4-2.1 探針A與探針B的比較---------------41
4-3 探針C之實驗結果--------------------43
第五章: 實驗結論-----------------------45
第六章、實驗部分-----------------------46
6-1 實驗器材與藥品---------------------47
6-2 細胞影像實驗-----------------------48
6-2.1 細胞之準備-----------------------48
6-2.2 探針之MCF-7細胞影像--------------48
6-3 有機合成與光譜資料-------------------49
第七章: 參考資料-----------------------61
附錄----------------------------------66

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