A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells.
The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.
Albarran
Brian
B
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
To
Richard
R
Stayton
Patrick S
PS
D000179:Acrylates / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D011992:Endosomes / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology; D015696:Gene Products, tat / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D046911:Macromolecular Substances / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D010799:Phycoerythrin / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry*; D054322:tat Gene Products, Human Immunodeficiency Virus
D000179:Acrylates; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D046911:Macromolecular Substances; D010455:Peptides; D011108:Polymers; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D010799:Phycoerythrin; D019809:Streptavidin; D000469:Alkaline Phosphatase; C036658:acrylic acid