title: Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells.
abstract: To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated.
authors.affiliation: Department of Human Physiology, Centre for Neuroscience, Flinders Medical Research Institute, Flinders University of South Australia, Adelaide, Australia. Shahram.Barati@post.flinders.edu.au
authors.forename: Shahram
authors.initials: S
authors.lastname: Barati
authors.forename: Fariba
authors.initials: F
authors.lastname: Chegini
authors.forename: Plinio
authors.initials: P
authors.lastname: Hurtado
authors.forename: Robert A
authors.initials: RA
authors.lastname: Rush
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D004268:DNA-Binding Proteins; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques / Q000639:trends; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013744:Tetanus Toxin / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D004167:Diphtheria Toxin; C052130:GAL4 protein, S cerevisiae; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013744:Tetanus Toxin; D014157:Transcription Factors; C040635:tetanus toxin fragment C