Protocol

Site-Specific Protein Labeling

Volume 1266 of the series Methods in Molecular Biology pp 81-92

Date:

2-Cyanobenzothiazole (CBT) Condensation for Site-Specific Labeling of Proteins at the Terminal Cysteine Residues

  • Lina CuiAffiliated withMolecular Imaging Program at Stanford, Departments of Radiology and Chemistry, School of Medicine, Stanford University Email author 
  • , Jianghong RaoAffiliated withMolecular Imaging Program at Stanford, Departments of Radiology and Chemistry, School of Medicine, Stanford University

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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 18F via CBT condensation.

Key words

Site-specific protein labeling Terminal cysteine 1,2-Aminothiol 2-Cyanobenzothiazole (CBT) Fluorescence labeling Radiolabeling (18F)