Gaur Reetika, Gupta Pradeep K, Goyal Anita, Wels Winfried, Singh Yogendra
Centre for Biochemical Technology, Mall Road, Near Jubilee Hall, 110 007, Delhi, India.
Biochem Biophys Res Commun. 2002 Oct 11;297(5):1121-7. doi: 10.1016/s0006-291x(02)02299-4.
Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.
基于受体介导的内吞作用的基因递送载体提供了一种有吸引力的长期解决方案，因为它们可能克服许多病毒基因递送系统固有的毒性和载量限制。炭疽毒素的保护性抗原成分与特定受体结合，并将致死因子或水肿因子递送至哺乳动物细胞的胞质溶胶中。致死因子（LF）的N端254个氨基酸（LF(1-254)）与保护性抗原（PA）结合，当与异源蛋白融合时，可将此类蛋白递送至胞质溶胶中。然而，到目前为止，尚未尝试使用炭疽毒素系统进行DNA的细胞内递送。在本研究中，炭疽毒素的LF(1-254)与GAL4蛋白的DNA结合结构域融合。融合蛋白（LF(254)-GAL4DBD）在溶液中既表现出PA结合活性，也表现出DNA结合活性。融合蛋白与含有报告基因（荧光素酶或绿色荧光蛋白）的质粒DNA以及PA形成的复合物将质粒DNA递送至COS-1细胞的胞质溶胶中。这些结果表明，炭疽毒素成分可作为一种非病毒系统，用于将DNA高效递送至哺乳动物细胞的胞质溶胶中。
