| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Tohru Sankyo Biomedical Laboratories, Senior Scientist, バイオメディカル研究所, 副主任研究員
KUME Akihiro Jichi Medical School, Faculty of Medicine, Assistant Professor, 医学部, 講師 (10264293)
OZAWA Keiya Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)
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| Research Abstract |
We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, β-galactosidase with the nuclear localization signal of SV40 (nβ-gal), within the cytoplasm, it was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor(G-CSFR). To liberate the nβ-gal portion from the fusion protein, we used a protease derived from clover yellow vein virus, Nia protease, whose recognition sequence was inserted between the G-CSFR and nβ-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescent staining using an anti-P-gal antibody. When expressed together with the Nia, β-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the Nia, indicating this protease is not cytotoxic. Based on these results, we constructed a dicistronic plasmid expressing an Rep protein fused with G-CSFR driven by the p5 promoter and the blasticidin S resistance gene under the translational control of the encephalomyocarditis virus internal ribosome entry site (IRES). Following transfection of 293 cells with this plasmid and the selection with blasticidin S, we isolated cell clones that could produce recombinant AAV after transfection with small Rep/Cap plasmid, Adenovirus helper plasmid, and Nia plasmid. The yield of rAAV was increased 10-fold when the Nia plasmid was cotransfected (about 105 particles/10-cm dish). However, the titer of rAAV produced by these cell lines gradually decreased as they were passaged. These results indicated the presence of a leaky nuclear transport of the chimeric protein even in the absence of the Nia protease.
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