Abstract
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.
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Acknowledgments
The authors wish to thank New England Biolabs and Donald. G. Comb, Jim Ellard, Richard Roberts, and Christopher Noren for their support and suggestions. We thank the Organic Division for peptide synthesis.
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Ghosh, I. et al. (2011). Site-Specific Protein Labeling by Intein-Mediated Protein Ligation. In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_6
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DOI: https://doi.org/10.1007/978-1-61737-967-3_6
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