title: C2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein p53 into tumor cells.
abstract: We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fahrer
authors.forename: Brigitte
authors.initials: B
authors.lastname: Schweitzer
authors.forename: Katja
authors.initials: K
authors.lastname: Fiedler
authors.forename: Torben
authors.initials: T
authors.lastname: Langer
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D001710:Biotin; D019809:Streptavidin