Gadi Jogeswar, Ruthala Kalyani, Kong Kyoung-Ah, Park Hyoung Woo, Kim Myoung Hee
Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Sodaemun-gu, Seoul, 120-752, South Korea.
Mol Biotechnol. 2009 May;42(1):41-8. doi: 10.1007/s12033-008-9119-7. Epub 2008 Nov 8.
Protein transduction domains (PTDs) have been shown to cross the biological cell membranes efficiently through a receptor and energy independent mechanism. Because of its ease in membrane transducing ability, PTDs could be used as a gene delivery vector. Since we already have shown that purified Hoxc8 homeoprotein has the ability to cross the cellular membrane, we analyzed the possibility of the third helix of the Hoxc8 homeodomain as a useful gene delivery vector. For that purpose, a 16-aa long synthetic oligopeptide Hoxc8 Protein Transduction Domain (HPTD) was chemically synthesized and then tested to see whether the HPTD could form a complex with DNA or not. Gel retardation analysis revealed that the HPTD interacts with plasmid DNA efficiently but failed to transfer the DNA into the cells. However, HPTD can enhance the efficiency of gene transfer in combination with Lipofectamine which doubled the gene transfer rate into COS-7 cells compared with the DNA/Lipofectamine control. An MTT assay indicated that the amount of HPTD used in the complex for the transfection did not show any cytotoxicty in COS-7 cells. The TEM studies showed compact particle formation in the presence of HPTD. These results indicate that the HPTD could be a good candidate adjuvant molecule to enhance the gene transfer efficiency of Lipofectamine in eukaryotic cells.
蛋白质转导结构域（PTDs）已被证明能通过一种不依赖受体和能量的机制有效穿过生物细胞膜。由于其膜转导能力简便易行，PTDs可被用作基因传递载体。既然我们已经证明纯化的Hoxc8同源蛋白具有穿过细胞膜的能力，我们分析了Hoxc8同源结构域的第三个螺旋作为一种有用的基因传递载体的可能性。为此，化学合成了一个16个氨基酸长的合成寡肽Hoxc8蛋白质转导结构域（HPTD），然后测试HPTD是否能与DNA形成复合物。凝胶阻滞分析表明，HPTD能有效与质粒DNA相互作用，但未能将DNA转入细胞。然而，与Lipofectamine联合使用时，HPTD可提高基因转移效率，与DNA/Lipofectamine对照组相比，转入COS-7细胞的基因转移率提高了一倍。MTT分析表明，转染复合物中使用的HPTD量在COS-7细胞中未显示任何细胞毒性。透射电镜研究表明，在有HPTD存在的情况下会形成致密颗粒。这些结果表明，HPTD可能是提高Lipofectamine在真核细胞中基因转移效率的良好辅助分子候选物。
