Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Biochim Biophys Acta. 2010 Dec;1798(12):2249-57. doi: 10.1016/j.bbamem.2010.02.003. Epub 2010 Feb 17.


Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell-Penetrating Peptides / biosynthesis
  • Cell-Penetrating Peptides / chemistry
  • Cell-Penetrating Peptides / genetics
  • Cell-Penetrating Peptides / isolation & purification
  • Cell-Penetrating Peptides / pharmacology
  • Chromatography, Affinity / methods
  • Endotoxins*
  • Escherichia coli
  • Gene Expression
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / pharmacology
  • HeLa Cells
  • Humans
  • Inteins
  • Recombinant Fusion Proteins* / biosynthesis
  • Recombinant Fusion Proteins* / chemistry
  • Recombinant Fusion Proteins* / genetics
  • Recombinant Fusion Proteins* / isolation & purification
  • Recombinant Fusion Proteins* / pharmacology


  • Cell-Penetrating Peptides
  • Endotoxins
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins