title: Genetically engineered clostridial C2 toxin as a novel delivery system for living mammalian cells.
abstract: The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a K(D)-value of approximately 0.75 muM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 47, D-89081 Ulm, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fahrer
authors.forename: Rainer
authors.initials: R
authors.lastname: Plunien
authors.forename: Ulrike
authors.initials: U
authors.lastname: Binder
authors.forename: Torben
authors.initials: T
authors.lastname: Langer
authors.forename: Hartmut
authors.initials: H
authors.lastname: Seliger
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D015151:Immunoblotting; D008264:Macrophages / Q000378:metabolism; D018613:Microscopy, Confocal; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
chemical_list: D005452:Fluoresceins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D001905:Botulinum Toxins