ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

Expression and Purification of ATP-dependent Human Lon Protease and Characterization of its Enzymatic Activity

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2+柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-(CBZ-L-alanyl-L-alanine amide)[(Z-AA)2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。

Human Lon protease is a homo-oligomeric ring-shaped ATP-dependent protease located in mitochondrial matrix.Recent researches demonstrated that Lon protease plays an important role in cellular homeostasis,mitochondrial protein quality control and metabolic regulation.Human Lon expression plasmid carrying a carboxyl-terminal hexahistidine tag was transformed into the E.coli Rosetta 2 strain and we optimized the induction condition for high expression.Ni2＋-NTA agarose was used to purify this his-tag Lon protease.The peptidase activity of Lon was measured by using peptide substrate Rhodamine 110,bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110],Casein and purified TFAM as substrates.The results showed that purified Lon retained the ATP-dependent enzymatic activity.