Effective delivery of engineered proteins into mitochondria is of great significance for developing efficient mitochondrial DNA editing tools and realizing accurate treatment of mitochondrial diseases. Here, the candidate genes, eGFP and Cas9, were engineered with different mitochondrial localization signal (MLS) sequences introduced at their up- or/and down-streams. The corresponding expression vectors for the engineered proteins were constructed respectively, and HEK293T cells were transfected with these vectors. The fluorescence colocalization and Western blotting assays were used to analyze the mitochondrial targeting presentation effect of different engineered proteins. The results demonstrated that the daul-MLS modification of the eGFP and Cas9 proteins significantly improved the efficiency of mitochondrial targeted presentation, compared with the engineered proteins with single MLS added. Hence, it is speculated that dual MLS strategy can enhance the mitochondrial targeting of engineered proteins, which lays a theoretical foundation for the future development of efficient mitochondrial DNA editing tools.
有效传递工程化改造的蛋白进入线粒体对开发高效的线粒体DNA编辑工具、实现线粒体疾病精准治疗具有重要意义。本研究选取eGFP和Cas9基因，在其上游或/和下游引入不同的线粒体定位信号(mitochondrial localization signal，MLS)序列，分别构建了相应的工程化蛋白表达载体。将不同表达载体转染HEK293T细胞后，利用荧光共定位实验和免疫印迹实验分析不同工程化蛋白的线粒体靶向性呈递效果。结果显示，相比单端添加MLS的eGFP和Cas9蛋白，双端MLS改造均显著提高了工程化蛋白的线粒体靶向性呈递效率。推测双MLS策略可增强工程化蛋白的线粒体靶向性，为以后开发高效的线粒体DNA编辑工具奠定了理论基础。.
Keywords: mitochondrial diseases; mitochondrial localization signal; mitochondrial targeting; mtDNA editing; protein engineering.