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

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 

Notes

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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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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