title: Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.
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.
authors.affiliation: Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang Fu Rd. Hsinchu, 30013 (Taiwan).
authors.forename: Hsiao-Chan
authors.initials: HC
authors.lastname: Wang
authors.forename: Ching-Ching
authors.initials: CC
authors.lastname: Yu
authors.forename: Chien-Fu
authors.initials: CF
authors.lastname: Liang
authors.forename: Li-De
authors.initials: LD
authors.lastname: Huang
authors.forename: Jih-Ru
authors.initials: JR
authors.lastname: Hwu
authors.forename: Chun-Cheng
authors.initials: CC
authors.lastname: Lin
mesh_terms: D052160:Benzothiazoles / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D058185:Magnetite Nanoparticles / Q000737:chemistry; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D009570:Nitriles / Q000737:chemistry*; D040081:Protein Array Analysis
chemical_list: C578355:2-cyanobenzothiazole; D052160:Benzothiazoles; D055767:Immobilized Proteins; D058185:Magnetite Nanoparticles; D058619:Maltose-Binding Proteins; D009570:Nitriles; D010450:Endopeptidases; C099488:TEV protease; D003545:Cysteine
keywords: click chemistry; cyanobenzothiazole; microarrays; protein immobilization; site-selective