Sending regulatory elements into the nuclei from medium.

本项目的目的是建立一种新的系统，该系统能够使用白喉毒素作为载体将生物活性蛋白质或酶引入细胞核。白喉毒素与哺乳动物细胞上的特异性受体结合，并且A片段到达细胞质以发挥其毒性。我们设计了一个含有白喉毒素B片段和T4核酸内切酶V的融合基因，并在大肠杆菌中进行了表达。杆菌产物用盐酸胍溶解，通过逐步透析复性，并通过连续色谱纯化。纯化的融合蛋白与白喉毒素受体的结合亲和力比天然毒素高约10倍。融合蛋白切口的封闭的环状质粒DNA，其预先暴露于紫外线的效率类似于核酸内切酶V。细胞系来源于着色性干皮病与融合蛋白孵育，并确定其计划外的DNA合成后，紫外线照射。与没有融合蛋白的对照细胞相比，与融合蛋白孵育的细胞显示出程序外DNA合成的轻微增加，而蛋白质对未用UV光照射的细胞没有影响。这些结果表明，融合蛋白到达细胞核，在那里它发挥酶活性。然而，需要进一步的研究来制造实际有用的分子，因为易位的效率太低而不能用于应用。

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.