Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant proteins expressed in E. coli using synthetic short peptides possessing a fluorescent moiety. This approach can be readily utilized for site-specific conjugation of a fluorophore to the C-terminus of a protein of interest, without the drawback of non-specific chemical labeling. This chapter also gives a general review of the critical parameters of intein-mediated cleavage and ligation reactions.
CITATION STYLE
Ghosh, I., Considine, N., Maunus, E., Sun, L., Zhang, A., Buswell, J., … Xu, M. Q. (2011). Site-Specific Protein Labeling by Intein-Mediated Protein Ligation. In Methods in Molecular Biology (Vol. 705, pp. 87–107). Humana Press Inc. https://doi.org/10.1007/978-1-61737-967-3_6
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