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Chembiochem. 2014 Apr 14;15(6):829-35. doi: 10.1002/cbic.201300800. Epub 2014 Mar 5.

Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.

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  • 1Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd. Hsinchu, 30013 (Taiwan).


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.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


click chemistry; cyanobenzothiazole; microarrays; protein immobilization; site-selective

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