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Methods Mol Biol. 2011;705:87-107. doi: 10.1007/978-1-61737-967-3_6.

Site-specific protein labeling by intein-mediated protein ligation.

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  • 1New England BioLabs, Ipswich, MA 01938, USA.


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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