定向进化后Cne PRP8蛋白质内含子的剪接机理研究  

Study on the splicing mechanism of Cne PRP8 intein with directional evolution

作　　者：李淑梦 李曼 孟清[1] LI Shu-meng;LI Man;MENG Qing(Institute of Biological Sciences and Biotechnology,Donghua Univresity,Shanghai 201620,China)

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《生物学杂志》2018年第5期8-12,17,共6页Journal of Biology

基　　金：国家自然科学基金项目(31570721);上海市科委公关项目(14521100700);上海市科委国际合作项目(14520720200);国家外专局高端外国专家项目(GDW20143100071)

摘　　要：蛋白质内含子(intein)介导的蛋白质剪接(protein splicing)为蛋白质工程的发展展现了无限的新希望。但是到目前为止蛋白质剪接机理尚未清楚,因而它极大地制约了蛋白质内含子及其介导的蛋白质剪接在蛋白质工程领域的广泛应用。以来自于真核生物核基因组中的蛋白质内含子Cne PRP8为研究对象,通过"随机突变-功能选择-定向进化"后,对进化后的蛋白质内含子Cne PRP8关键部位氨基酸进行突变、恢复突变,试图揭示Cne PRP8蛋白质内含子的剪接机理。研究结果表明,Cne PRP8蛋白质内含子的剪接仍然与其1位和正1位相关,但其剪接反应还与其52位、68位、114位和146位的氨基酸相关,推测该位氨基酸的突变改变了剪接活性中心的空间构象,从而影响了蛋白质内含子的剪接。这一研究揭示了真核生物蛋白质内含子的剪接过程不仅与其1位和正1位等氨基酸残基相关,也与其他位点氨基酸密切相关,仍需进一步探索与研究。Intein-mediated protein splicing offers many promises in protein engineering. However, the mechanisms of protein splicing is still unclear, which greatly limits the general application of protein splicing in protein engineering. Cne PRP8 intein from a eukaryotic genome had been improved through directed evolution consisting of random mutagenesis followed with functional selection. This improved Cne PRP8 intein will be subjected to site-specific mutations of amino acid residues at critical locations, followed with restoration mutation, and mutational studies of extein amino acid residues at the splice junctions, in order to discover new mechanisms of the protein splicing. The results show that the splicing of eukaryotic protein intein is still related to its one site and its positive one site, but for the intein of Cne PRP8 protein, its splicing efficiency is related to its the 52 th site, 68 th site,114 th site and 146 th site amino mutations, and the mutations of its amino acids change the polarity, thus affecting the splicing of protein introns. This study not only consolidates the mechanism of protein intron splicing, but also reveals the evolutionary pathway of protein introns in eukaryotes.

关 键 词：蛋白质内含子 蛋白质剪接 蛋白质剪接机理 

分 类 号：Q51[生物学—生物化学] Q75