Cellular translocation of proteins by transportan

Proteins with molecular masses ranging from 30 kDa (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein–peptide constructs to penetrate into Bowes melanoma, BRL, and COS‐7 cells. After 0.5–3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS‐7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.