Work published in Science (285, 1569–1572, 1999) may open new possibilities for both protein replacement therapies and drug delivery in general. Until now, the plasma membrane has prevented drugs larger than 600 Da in size from entering cells, making most intracellular targets inaccessible to bulky protein treatments. Now Howard Hughes Investigator Steven Dowdy and his colleagues at the Washington University School of Medicine in St. Louis have successfully transferred into mice a protein almost 200 times larger than the size threshold. This feat was accomplished by tagging a protein of interest with a unique 11 amino-acid protein transduction domain (PTD) from HIV Tat protein and subsequently denaturing the resultant fusion with urea. According to Dowdy, denaturation uncovers hydrophobic residues that help the fusions traverse cell membranes "like parting the Red Sea." Previous in vitro studies by his group had already proved the approach successful for 60 proteins (ranging in size from 15 kDa to 120 kDa). But the results obtained in mice were better than they could have dreamed: "We were blown away," says Dowdy. Fusions with fluorescein and β-galactosidase injected into the peritoneum penetrated all tissues, including the brain (see inset), in under 25 minutes. What's more, once inside the cell, β-galactosidase both refolded and regained its activity. "The system also has obvious utility for basic studies of protein function," says Dowdy. "Over 400 laboratories around the world are now using it." Currently, the technology has been licensed to Life Technologies (Rockville, MD) for basic research applications and to IDUN Pharmaceuticals (La Jolla, CA) for therapeutics.