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.