Sending regulatory elements into the nuclei from medium.

点击翻译按钮获取中文摘要

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.