The mammalian cell membrane is refractory to most proteins and peptides. Until recently, the most widely used methods to introduce proteins and peptides into cells involved microinjection or electrporation. Microinjection requires specialized equipment and technical skill and can be performed only in one cell at a time. Electroporation is more effective but kills a large proportion of cells. However, recently there has been a growing range of products in the market which facilitate the delivery of proteins to the nucleus and the cytosol in a simple and effective fashion.
The Chariot reagent by Active Motif is one such reagent for introducing proteins into cells. It is based on a short synthetic signaling peptide called Pep-1. The most important advantage of the Chariot reagent is that it is a non-covalent carrier of peptides and proteins into cells. Most of the protein delivery systems require covelent coupling to the bioactive peptide (or protein). Such covalent conjugation with the carrier may perturb the bioactive conformation of the cargo peptide and cause partial loss of bioactivity. However, the Chariot reagent circumvents this obstacle by forming non-covalent hydrophobic interactions with its cargo protein. Furthermore, the Chariot-macromolecule complex stabilizes the protein and protects it from the endosomal pathway thereby ensuing a high degree of internalization of the cargo protein.
The other main advantage of the Chariot reagent is that the time required for introducing the protein into cells is much shorter than conventional transfection methods. The reagent is mixed with the peptide (or proteins) for 30 minutes in buffer to allow the association of Chariot to the peptide. This complex is then added to the cells. Once inside the cell the Chariot and its cargo rapidly dissociate and the protein activity can be assayed after just a few hours. The Chariot reagent works for all cell types tested in the lab including endothelial cells and primary human fibroblasts, which are notoriously difficult to transfect. The reagent is non-cytotoxic and has been found to be highly efficient for the delivery of small bioactive peptides in our laboratory.
The Chariot reagent does have a few limitations. One of the methods for improving protein delivery is to dilute the Chariot reagent in DMSO before adding it to the cargo protein. However the percentage of DMSO requited for such enhanced delivery is about 40-60%, which causes extensive cell death in most cell types. Another drawback is that the Chariot-protein complex dissociates inside cells releasing its cargo very rapidly. Hence, the reagent is not suitable for drug delivery studies, which require a slower and more sustained release of therapeutic peptides and proteins. Despite these limitations it is an excellent, non-invasive transfection reagent for efficient protein delivery into mammalian cells.
Piyali Dasgupta, Ph.D.
Post-Doctoral Fellow
Moffit Cancer Center