| Project/Area Number |
04044156
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Kurume University |
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Principal Investigator |
MEKADA Eisuke Institute of Life Science Kurume University Professor, 分子生命科学研究所, 教授 (20135742)
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| Co-Investigator(Kenkyū-buntansha) |
MURPHY Johon R. Medical Center Boston University Professor, メディカルセンター, 教授
YAMAIZUMI Masaru Institute of Molecular Embryology and Genetics Kumamoto University Professor, 医学部附属遺伝医学研究所, 教授 (70107093)
YONEDA Yoshihiro Institute for Molecular and Cellular Biology Osaka University Assistant Professo, 細胞工学センター, 助教授 (80191667)
IWAMOTO Ryo Institute of Life Science Kurume University Research Associate, 分子生命科学研究所, 助手 (10213323)
TSUNEOKA Makoto Institute of Life Science Kurume University Assistant Professor, 分子生命科学研究所, 講師 (50197745)
JOHON R.Murp ボストン大学, メディカルセンター, 教授
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥10,200,000 (Direct Cost: ¥10,200,000)
Fiscal Year 1993: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1992: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | Diphtheria toxin / Endonuclease / Xeroderma pigmentosum / Fusion protein / FuragmentB |
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| Research Abstract |
The aim of this project is to establish a new system which enables to introduce biologically active proteins or an enzymes into nuclei using diphtheria toxin as a carrier. Diphtheria toxin binds to the specific receptor on mammalian cells and the A fragment reaches to the cytoplasm to exert its toxicity. We have designed to replace the A fragment with another protein which carries naturally or artificially a nuclear-localizing signal, and to test whether the constructed fusion protein, or a part of the protein, reaches to nuclei and acts there when the protein is added to the culture medium.
A fusion gene comprising diphtheria toxin B fragment and T4 endonuclease V was constructed and it was expressed into E. coli. The product was dissolved with guanidium-HCL, renatured by step-wise dialysis and purified by sequential chromatography. The purified fusion protein bound to the diphtheria toxin receptor with an affinity about 10 times higher than that of native toxin. The fusion protein nicked a closed circular plasmid DNA which was pre-exposed with UV light with an efficiency similar to that of endonuclease V. Cell lines derived from Xeroderma pigmentosum were incubated with the fusion protein and whose unscheduled DNA synthesis after UV-light irradiation was determined. Cells incubated with the fusion protein showed a slight increase of the unscheduled DNA synthesis compared with control cells without the fusion protein, while the protein had no effect on cells not irradiated with UV light. These results indicate that the fusion protein reaches to the nuclei where it acts enzymatic activity. However, further investigation will be required to make actually useful molecules, because the efficiency of translocation is too low to use for the application.
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