title: A gene delivery system based on the N-terminal domain of human topoisomerase I.
abstract: The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in charge and has DNA binding activity, without DNA sequence and topological specificity. Here, a fusion protein (6 x His-PTD-TopoN) containing a hexahistidine (6 x His) tag, a membrane penetration domain and TopoN (amino acid 3-200) was designed and developed. The protein can bind to different sizes (3.0-8.0 kb) and forms (circular and linear) of DNA and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in vitro. Maintaining the hexahistidine tag increased the protein's transfection efficiency in COS7 African green monkey kidney cells and simplified the purification process. The plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and temperature-dependent manners. The results indicate that the binding ability of TopoN is very useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without removal of the hexahistidine tag.
authors.affiliation: Laboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan.
authors.forename: Yi-An
authors.initials: YA
authors.lastname: Chen
authors.forename: Hsiao-Che
authors.initials: HC
authors.lastname: Kuo
authors.forename: Young-Mao
authors.initials: YM
authors.lastname: Chen
authors.forename: Shin-Yi
authors.initials: SY
authors.lastname: Huang
authors.forename: Yu-Ru
authors.initials: YR
authors.lastname: Liu
authors.forename: Su-Ching
authors.initials: SC
authors.lastname: Lin
authors.forename: Huey-Lang
authors.initials: HL
authors.lastname: Yang
authors.forename: Tzong-Yueh
authors.initials: TY
authors.lastname: Chen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human