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

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。

關鍵字

蛋白質固化；位向選擇性修飾； 2-氰基苯並噻唑縮合反應

並列摘要

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.

並列關鍵字

Protein Immobilization ； Site-Specific Modification ； CBT condensation

參考文獻

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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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