Abstract
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.