Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I.
Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51-200, 271-375, 422-596, and 651-696 of the human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy.
Chen
T Y
TY
Institute of Genetics, School of Life Science, National Yang-Ming University, Taipei, Taiwan, ROC.
Hsu
C T
CT
Chang
K H
KH
Ting
C Y
CY
Whang-Peng
J
J
Hui
C F
CF
Hwang
J
J
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011550:Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D005098:Exotoxins; D005982:Glutathione Transferase; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A