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.
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Acknowledgement
This work was funded by a grant from NIGMS (R01GM086196-01), the IDEA award from Department of Defense Breast Cancer Research Program (W81XWH-09-1-0057) and the NCI ICMIC at Stanford (1P50CA114747-06).
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Cui, L., Rao, J. (2015). 2-Cyanobenzothiazole (CBT) Condensation for Site-Specific Labeling of Proteins at the Terminal Cysteine Residues. In: Gautier, A., Hinner, M. (eds) Site-Specific Protein Labeling. Methods in Molecular Biology, vol 1266. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2272-7_5
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DOI: https://doi.org/10.1007/978-1-4939-2272-7_5
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