title: 2-Cyanobenzothiazole (CBT) condensation for site-specific labeling of proteins at the terminal cysteine residues.
abstract: Site specificity is pivotal in obtaining homogeneously labeled proteins without batch-to-batch variations. More importantly, precisely controlled modification at specific sites avoids potential pitfalls that could otherwise interfere with protein folding, structure, and function. Inspired by the chemical synthesis of D-luciferin, we have developed an efficient strategy (second-order rate constant k 2 = 9.2 M(-1) s(-1)) for labeling of proteins containing 1,2-aminothiol via reaction with 2-cyanobenzothiazole (CBT). In addition, the CBT condensation enjoys the convenience of protein engineering, as production of N-terminal cysteine-containing proteins has been well developed for native chemical ligation. This protocol describes the preparation of Renilla luciferase (rLuc) with 1,2-aminothiol at either its N- or C-terminus, and site-specific labeling of rLuc with fluorescein or (18)F via CBT condensation.
authors.affiliation: Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, School of Medicine, Stanford University, Lucas Center Rm P093, 1201 Welch Road, Stanford, CA, 94305-5484, USA, linacui@stanford.edu.
authors.forename: Lina
authors.initials: L
authors.lastname: Cui
authors.forename: Jianghong
authors.initials: J
authors.lastname: Rao
mesh_terms: D052160:Benzothiazoles / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: C578355:2-cyanobenzothiazole; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D009570:Nitriles; D011993:Recombinant Fusion Proteins; D049410:Luciferases, Renilla; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine