Heterologous Gene Expression in E.coli pp 87-107

Part of the Methods in Molecular Biology book series (MIMB, volume 705)

Site-Specific Protein Labeling by Intein-Mediated Protein Ligation

  • Inca Ghosh
  • Nancy Considine
  • Elissa Maunus
  • Luo Sun
  • Aihua Zhang
  • John Buswell
  • Thomas C. EvansJr
  • Ming-Qun Xu
Protocol

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.

Key words

Intein-mediated protein ligation expressed protein ligation intein protein labeling 

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Inca Ghosh
    • 1
  • Nancy Considine
    • 1
  • Elissa Maunus
    • 1
  • Luo Sun
    • 1
  • Aihua Zhang
    • 1
  • John Buswell
    • 1
  • Thomas C. EvansJr
    • 2
  • Ming-Qun Xu
    • 3
  1. 1.New England BioLabsIpswichUSA
  2. 2.DNA Enzymes DivisionNew England BioLabsIpswichUSA
  3. 3.Chemical Biology DivisionNew England BioLabsIpswichUSA

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