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- Title
Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.
- Authors
Wang, Hsiao-Chan; Yu, Ching-Ching; Liang, Chien-Fu; Huang, Li-De; Hwu, Jih-Ru; Lin, Chun-Cheng
- Abstract
We described a rapid site-selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT-solid support to immobilize the protein. According to microarray analysis, we found that glutathione S-transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N- and C-terminally immobilized maltose-binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT-Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site-selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.
- Publication
Chembiochem : a European journal of chemical biology, 2014, Vol 15, Issue 6, p829
- ISSN
1439-7633
- Publication type
Journal Article
- DOI
10.1002/cbic.201300800