This proposal outlines the development of a system to introduce peptides into mammalian tissue culture cells in a rapid and efficient manner. This system utilize a 16 amino acid peptide, derived from the Drosophilia Antennapedia homeoprotein, that is translocated across neuronal membranes when incubated extracellularly. Internalization of this peptide does not appear to be mediated by endocytosis or require a receptor, suggesting that it may be useful in a variety of cell types. A prokaryotic expression vector win he constructed for cost-efficient synthesis and purification of Antennapedia fusion peptides. Translocation of peptide fusions of different sizes will be tested to determine the steric constraints of this system. The translocation of these peptides will also be tested with many common tissue culture cell lines to prove the utility of this technology. Unlike techniques requiring direct injection of peptides into cells, introduction of this peptide vector into cells is rapid and efficient. Once internalized, this peptide is found in both the cytoplasm and nucleus. This technology has a wide variety of applications, including the delivery of inhibitory or therapeutic peptides, the delivery of oligonucleotides, and gene therapy. PROPOSED COMMERCIAL APPLICATION: Phase I research will result in a prokaryotic plasmid vector that allows simple expression and purification of Antennapedia peptide fusions. These peptides can be used to introduce peptides or oligonucleotides into cells quickly and efficiently. The Antennapedia technology can he applied to research in many disciplines, including gene transfer and gene therapy.
Thesaurus Terms:DNA binding protein, biotechnology, chimeric protein, transfection, transfection vector Drosophilidae, method development, oligonucleotide, peptide structure, protein sequence, protein transport PC12 cell, prokaryote, protein purification, tissue /cell cultureNational Institute of Mental Health (NIMH)
