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Site‐Selective Protein Immobilization through 2‐Cyanobenzothiazole‐Cysteine Condensation
Author(s) -
Wang HsiaoChan,
Yu ChingChing,
Liang ChienFu,
Huang LiDe,
Hwu JihRu,
Lin ChunCheng
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300800
Subject(s) - chemistry , cysteine , n terminus , tobacco etch virus , peptide , biochemistry , c terminus , amino acid , peptide sequence , enzyme , biology , plant virus , virus , potyvirus , virology , gene
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.