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  • Title: Site-specific protein labeling by intein-mediated protein ligation.
    Author: Ghosh I, Considine N, Maunus E, Sun L, Zhang A, Buswell J, Evans TC, Xu MQ.
    Journal: Methods Mol Biol; 2011; 705():87-107. PubMed ID: 21125382.
    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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