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

Abstract Expressed protein ligation(EPL) is a useful method for the native chemical ligationof proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteinsof 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 thioesterof EGFP (EGFP-SR) was prepared. Optimization of the ligationof EGFP-SR with arginine 12-mer (R12) produced the fusion proteinin 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 ligationwith the R12 peptide. Fusion proteinsof EGFP with R12 and the d -isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion proteindirectly expressed in E scherichia coli .