The delivery of biomolecules by cell penetrating peptides (CPPs) is an innovative therapeutic strategy. However delivery efficiency is hindered by the entrapment of CPPs in vesicles, degradation, or recycling out of cells, which limits their delivery into the cell cytoplasm and nucleus. To overcome these barriers, we investigated a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA, residues 253-412) that is able to exit vesicular compartments. A series of CPP-ETA(253-412) fusion proteins were constructed, expressed, and purified. Confocal microscopy and flow cytometry confirmed the internalization at 37oC of constructs containing CPPs (poly-arginine or TAT). In addition, constructs containing CPP-ETA(253-412)-eGFP were shown to relocate from endosomes to the cytosol. CPP-ETA(253-412) constructs were also able to act as carriers of DNA cargos facilitating their delivery to the cytosol. The ETA(253-412) translocation domain may prove useful for the intracellular delivery of drugs, protein therapeutics, siRNA delivery, and vaccine formulations.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/18890 |
| Date | 15 February 2010 |
| Creators | Broad, Amaalia |
| Contributors | Gariepy, Jean |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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