2-氰基苯並噻唑縮合反應於位向選擇性蛋白質固化之應用

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研究生：	王筱嬋 Wang, Hsiao-Chan
論文名稱：	2-氰基苯並噻唑縮合反應於位向選擇性蛋白質固化之應用 The Application of 2-Cyanobenzothiazole Condensation for Site-Selective Protein Immobilization
指導教授：	林俊成
口試委員:	林伯樵陳貴通林俊成
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	138
中文關鍵詞：	蛋白質固化、位向選擇性修飾、2-氰基苯並噻唑縮合反應
外文關鍵詞：	Protein Immobilization, Site-Specific Modification, CBT condensation
相關次數：	點閱：2 下載：0
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蛋白質固化是將目標蛋白質與固態載體經由適當的反應進行連結，使蛋白質能夠固定於固態載體表面並進行後續應用。在本論文中，利用2-cyanobenzothiazole（CBT）與1,2-aminothiol在水相溶液中進行快速縮合反應的特性，發展出具有位向選擇性的蛋白質固化製程。在固態載體的選擇上，玻片以及本實驗室已開發成熟的磁性奈米粒子，皆能夠成功地作為蛋白質位向專一性固化的載體。在蛋白質微陣列晶片的製程中，利用丙炔胺（propargylamine）修飾市售的N-hydroxysuccinimide（NHS）玻片得到參鍵表面修飾玻片，再透過點擊反應（Cu(I)-catalyzed alkyne/azide cycloaddition，CuAAC，亦稱 click chemistry）將末端修飾疊氮基之 CBT 衍生物裝配於玻片表面。CBT 功能化磁性奈米粒子的製程則是利用反應性較高的醯氯功能化磁性奈米粒子與帶有胺基之 CBT 衍生物進行親核性反應而得。
　　結合蛋白質純化表現系統 IMPACTTM-CN system 以及菸草鑲嵌病毒蛋白水解酶，我們能夠選擇性地經由化學或是酵素方式於蛋白質 N 端位或是 C 端位建構 1,2-aminothiol，並以此官能基進行蛋白質位向專一性固化。除了成功地固化綠色螢光蛋白之外，我們利用蛋白質晶片分析的結果顯示，以 N 端位固化的麩胺基硫轉移酵素較 C 端位進行固化具有較佳的活性；經由 N 或 C 端進行固化後的麥芽糖結合蛋白，其活性則差異不大。而以 N 端位固化於磁性奈米粒子上的菸草鑲嵌病毒蛋白水解酶其活性較以C端固化的方式佳。
　　在此我們成功地利用 CBT 與 1,2-aminothiol 的反應並結合IMPACTTM-CN system 以及菸草鑲嵌病毒蛋白水解酶，快速地製備出以位向選擇性固化樣品之蛋白質晶片以及磁性奈米粒子，並探討蛋白質固化位向與固化後活性間的關聯性。

Recently, protein immobilization has received considerable attention. In general, proteins are attached on solid supports through biocompatible reactions. In this thesis, a rapid and mild condensation reaction between 2-cyanobenzothiazole (CBT) and terminal 1,2-aminothiol was applied to achieve site-specific protein immobilization. Glass slides and magnetic nanoparticles (MNPs) were chosen as ideal solid supports to demonstrate the concept. The azido-CBT derivative was immobilized on alkynated glass slides through Cu(I)-catalyzed alkyne/azide cycloaddition (CuAAC, click chemistry) to prepared CBT-functionalized glass slides. The CBT-functionalized magnetic nanoparticles were prepared by amide bond formation between amino-CBT derivative and activated acylchloride group on MNPs.
By combination of IMPACTTM-CN system and tobacco etch virus protease (TEVp), the terminal 1,2-aminothiol was generated either at the N-terminal or C-terminal of protein of interest and reacted with the CBT-solid supports to achieve the immobilization of protein. According to microarray data analysis, we found that the glutathione S-transferase (GST) immobilized from its N-terminal retained higher substrate binding activity than that from C-terminal; whereas there were no differences in activities between neither N-terminal nor C-terminal immobilized maltose-binding protein (MBP). It was also observed that the immobilization of TEVp through N-terminal preserved higher activity than immobilization through C-terminal.
The success of utilizing CBT condensation reaction and easily constructing terminal 1,2-aminothiol by IMPACTTM-CN system and TEVp makes possibility of the developed method for alternatively site-specific protein immobilization on glass slides and nanoparticles. Furthermore, it is also demonstrated that the orientations of protein are crucial for its activity after being immobilized.

中文摘要..........................................................................................................i
Abstract    ..........................................................................................................iii
目錄.................................................................................................................v
圖目錄............................................................................................................xi
表目錄..........................................................................................................xiv
流程目錄.......................................................................................................xv
縮寫表..........................................................................................................xvi
第一章　序論.................................................................................................1
1 微陣列晶片.............................................................................................1
1.1 微陣列晶片的發展.........................................................................2
1.2 蛋白質微陣列晶片.........................................................................3
1.3 蛋白質固化方式.............................................................................6
1.3.1 隨機位向固化策略................................................................8
1.3.1.1 非共價性鍵結固化方式..........................................    ....8
1.3.1.2 共價性鍵結固化方式...................................................9
1.3.1.2.1 胺基反應............................................................10
1.3.1.2.2 硫醇基反應........................................................11
1.3.1.2.3 酸基反應    .........................................................13
1.3.1.2.4 環氧基反應........................................................14
1.3.1.2.5 光化學反應........................................................14
1.3.2 位向專一性固化策略    .........................................................16
1.3.2.1 共價性鍵結固化方式.................................................17
1.3.2.1.1 Diels-Alder Cycloaddtion...................................17
1.3.2.1.2 Staudinger ligation..............................................18
1.3.2.1.3 Click Chemistry...................................................19
1.3.2.1.4 Native chemical ligation    .....................................21
1.3.2.2 親和性結合固化方式.................................................25
1.3.2.2.1 His-Tag 與 Ni-NTA 系統................................25
1.3.2.2.2 Avidin 與 Biotin 系統......................................26
1.3.2.2.3 其他系統............................................................27
2 2-cyanobenzothiazole（CBT）與 1,2-aminothiol 縮合反應..................27
3 研究動機與目的....................................................................................32
第二章　實驗結果與討論...........................................................................33
1 蛋白質基因選殖與表達純化................................................................33
1.1 建構目標蛋白質基因之重組載體...............................................33
1.1.1 具菸草鑲嵌病毒蛋白水解酶辨識位之麥芽糖結合蛋白（T-MBP）.........................................................................................35
1.1.1.1 寡核酸引子之設計 ( Primer Designing )..................35
1.1.1.2 聚合酶鏈鎖反應 ( Polymerase Chain Reaction )......36
1.1.1.3 pGEM®-T Easy Vector 與插入段之黏接    ................38
1.1.1.4 質體 pTXB1 與插入段之黏接    ..............................40
1.1.2 具菸草鑲嵌病毒蛋白水解酶辨識位之綠色螢光蛋白（T-eGFP）    .....................................................................................41
1.1.2.1 寡核酸引子之設計（Primer Designing）....................41
1.1.2.2 聚合酶鏈鎖反應（Polymerase Chain Reaction）........42
1.1.2.3 pGEM®-T Easy Vector 與插入段之黏接................    ..43
1.1.2.4 質體 pTXB1 與插入段之黏接.................................44
1.1.3 具菸草鑲嵌病毒蛋白水解酶辨識位之麩胺基硫轉移酵素（T-GST）..........................................................................................44
1.1.3.1 寡核酸引子之設計（Primer Designing）.............    ..44
1.1.3.2 聚合酶鏈鎖反應 ( Polymerase Chain Reaction )    ..45
1.1.3.3 pGEM®-T Easy Vector 與插入段之黏接................    ..46
1.1.3.4 質體 pTXB1 與插入段之黏接    ..............................47
1.1.4 帶有自身辨識位之菸草鑲嵌病毒蛋白水解酶（T-TEVp）........................................................................................48
1.1.4.1 寡核酸引子之設計（Primer Designing）....................48
1.1.4.2 聚合酶鏈鎖反應 ( Polymerase Chain Reaction )    ..49
1.1.4.3 pGEM®-T Easy Vector 與插入段之黏接    ................50
1.1.4.4 質體 pTXB1 與插入段之黏接    ..............................50
1.2 重組目標蛋白質之誘導與純化    ..................................................50
1.2.1 帶有菸草鑲嵌病毒蛋白水解酶辨識位之麥芽糖結合蛋白（T-MBP）.........................................................................................51
1.2.1.1 誘導基因產物之表現    ............................................51
1.2.1.2 重組麥芽糖結合蛋白之純化    .....................................53
1.2.1.3 重組麥芽糖結合蛋白之 SDS – PAGE 膠體電泳分析    ............................................................................................54
1.2.2 帶有菸草鑲嵌病毒蛋白水解酶辨識位之綠色螢光蛋白（T-eGFP）    .....................................................................................56
1.2.2.1 誘導基因產物之表現    ............................................56
1.2.2.2 重組綠色螢光蛋白之純化    .....................................58
1.2.2.3 重組綠色螢光蛋白之 SDS – PAGE 膠體電泳分析.................................................................................................58
1.2.3 具菸草鑲嵌病毒蛋白水解酶辨識位之麩胺基硫轉移酵素（T-GST）..........................................................................................59
1.2.3.1 誘導基因產物之表現.................................................59
1.2.3.2 重組麩胺基硫轉移酵素之純化.................................61
1.2.3.3 重組麩胺基硫轉移酵素之 SDS – PAGE 膠體電泳分析    ............................................................................................61
1.2.4 帶有自身辨識位之菸草鑲嵌病毒蛋白水解酶（T-TEVp）.62
1.2.4.1 誘導基因產物之表現.................................................62
1.2.4.2 重組菸草鑲嵌病毒蛋白水解酶之純化    ................63
1.2.4.3 重組菸草鑲嵌病毒蛋白水解酶之 SDS – PAGE 膠體電泳分析.................................................................................63
1.2.5 引入菸草鑲嵌病毒蛋白水解酶辨識位後之目標蛋白質性質.......................................................................................................64
1.3 建構 N 端位 1,2-aminothiol......................................................66
1.3.1 菸草鑲嵌病毒蛋白水解酶（TEVp）之基因重組與表達純化.......................................................................................................66
1.3.1.1 寡核酸引子之設計 ( Primer Designing )..................68
1.3.1.2聚合酶鏈鎖反應 ( Polymerase Chain Reaction )........68
1.3.1.3 pGEM®-T Easy Vector 與插入段之黏接    ................69
1.3.1.4 質體 pTXB1 與插入段之黏接    ..............................70
1.3.1.5 菸草鑲嵌病毒蛋白水解酶之純化.............................70
1.3.1.6 菸草鑲嵌病毒蛋白水解酶之 SDS – PAGE 膠體電泳分析.........................................................................................71
1.3.2 利用 TEVp 水解反應修飾目標蛋白質............................71
1.4 建構 C 端位1,2-aminothiol.........................................................72
1.4.1 合成 di-Cysteine.................................................................72
1.4.2 利用 di-Cysteine 修飾目標蛋白質...................................73
1.5 目標蛋白質上1,2-aminothiol官能基之鑑定    ..............................73
2 透過CBT縮合反應製備蛋白質晶片    ..................................................77
2.1 CBT 修飾玻片的製備..................................................................77
2.2 CBT 修飾玻片的製程優化    .........................................................81
2.2.1 表面修飾反應時間..............................................................81
2.2.2 表面修飾的反應濃度..........................................................83
2.2.3 蛋白質固化反應時間    .........................................................84
2.2.4 蛋白質點樣濃度    ................................................................85
2.3 蛋白質固化於CBT 修飾玻片之位向與活性作用關聯之探討    ................................................................................................................86
3 TEVp固化於磁性奈米粒子（TEVp@MNP）    .....................................92
3.1 製備 CBT 功能化磁性奈米粒子...............................................96
3.2 蛋白質固化於 CBT 功能化磁性奈米粒子之位向與活性作用關聯之探討    ............................................................................................98
Chapter 3.  Experimental Material............................................................101
1 General Procedures and Materials.........................................................101
2 Synthesis................................................................................................101
2.1 Synthesis of di-Cysteine compound (2).......................................101
2.2 Synthesis of azido cyanobenzothiazole derivative (4)    ..............103
2.3 Synthesis of FITC-CBT (5)    .......................................................104
2.4 Synthesis of amino cyanobenzothiazole derivative (8)    ..............105
2.5 Synthesis of azide-terminated cyanobenzothiazole derivative (10)........................................................................................................106
2.6 Synthesis of GSH-Cy3.................................................................107
3 Construction of Expression Plasmids....................................................107
4 Protein Overexpression and Preparation of N-/C-terminal Cysteine Protein.........................................................................................................109
5 In vitro Protein Labeling by CBT Condensation Reaction....................110
6 Fabrication of Funcaionalized Slides....................................................110
6.1 Preparation of Alkyne-functionalized Slide.................................110
6.2 Preparation of CBT-functioned Slide    ..........................................111
6.3 Protein Spotting............................................................................111
7 Protein Microarray Analysis..................................................................111
8 Site-selectively Immobilization of TEVp on Magnetic Nanoparticles    .112
8.1 Preparation of CBT-functionalized Magnetic Nanoparticles (CBT@MNP)........................................................................................112
8.2 Protein Immobilization on CBT@MNP.......................................113
8.3 TEVp Activity Assay...................................................................113
第四章　結論.............................................................................................116
參考文獻.....................................................................................................118

                                

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