Heterologous Gene Expression in E.coli

Volume 705 of the series Methods in Molecular Biology pp 87-107


Site-Specific Protein Labeling by Intein-Mediated Protein Ligation

  • Inca GhoshAffiliated withNew England BioLabs
  • , Nancy ConsidineAffiliated withNew England BioLabs
  • , Elissa MaunusAffiliated withNew England BioLabs
  • , Luo SunAffiliated withNew England BioLabs
  • , Aihua ZhangAffiliated withNew England BioLabs
  • , John BuswellAffiliated withNew England BioLabs
  • , Thomas C. EvansJrAffiliated withDNA Enzymes Division, New England BioLabs
  • , Ming-Qun XuAffiliated withChemical Biology Division, New England BioLabs Email author 

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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.

Key words

Intein-mediated protein ligation expressed protein ligation intein protein labeling