Abstract
BackgroundThe cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47–57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes.ResultsBy confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs.ConclusionThis study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells.
Highlights
The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo
Characterization of the internalization of a novel TATstreptavidin (TAT-SA) constructs The structure of TAT-streptavidin (TAT-SA) has been previously described [10] as a tetrameric fusion protein, in which the TAT47–57 peptide has been attached to the N-terminus of each streptavidin monomer
Biotins or biotinylated molecules are located into binding pockets of SA
Summary
The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. BMC Biotechnology 2007, 7:1 http://www.biomedcentral.com/1472-6750/7/1 strategies to overcome the cell membrane barrier have been proposed, including electroporation, microinjection, viral vectors, liposome encapsulation and receptormediated endocytosis. These methods have, been plagued by low delivery efficiencies and, to some extent, increased cellular toxicity. Occurring short cell-penetrating peptides (CPPs) derived from viral, insect or mammalian proteins, have attracted considerable interest in the field of drug delivery for their ability to direct cellular uptake through active transport mechanisms. CPPs have been used to treat preclinical models of human disease [3,4]
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