Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide

Carrier peptides offer new opportunities to overcome problems in cellular drug delivery. Their objectives are improved cellular uptake or permeation of biological membranes, which are important pharmacokinetic features for the cellular distribution of therapeutics. Previously, human calcitonin (hCT) and selected C-terminal hCT fragments have been shown to be internalized and to permeate the epithelium of the nasal mucosa. To assess the potential of hCT-derived carrier peptides for cellular internalization of a model protein we fused enhanced green fluorescent protein (EGFP) and the [C(8)]hCT8-32 fragment by using expressed protein ligation (EPL). EGFP thioester was obtained by intein-mediated purification with an affinity chitin-binding tag (the IMPACT system, based on protein splicing). Internalization of EGFP-[C(8)]hCT8-32 by excised bovine nasal mucosa was monitored by confocal laser scanning microscopy. This novel conjugate displayed internalization into some sectors of the mucosa, whereas EGFP itself was not capable of translocation. Thus, we demonstrate successful internalization of a model protein through ligation to an hCT-derived carrier peptide, which has potential for the delivery of therapeutics. At this point the respective mechanism of translocation is unknown.