Expression, Purification and Characterization of Critical Domains of Munc13-1  

作　　者：Cong MA Hai HOU Wei TIAN Tao XU 

机构地区：[1]Joint Laboratory of Huazhong University of Science and Technology and Institute of Biophysics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074. China. [2]National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第8期617-623,共7页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China （30470448, 30130230）, and the Major State Basic Research Program of China （2004CB720000）

摘　　要：Munc13-1 is an essential component of synaptic vesicle releasing machinery. Three rat Munc 13-1 constructs were rationally designed based on homology and function, overexpressed in Escherichia coli, and purified to homogeneity with a final yield higher than 2 ~tg/ml of cell culture. The purified Munc 13-1 recombinant proteins had distinct oligomeric states, monodispersity and homogeneity properties. Their secondary structural contents were analyzed by the circular dichroism method, and the sedimentation coefficients of these recombinant proteins were measured by analytical ultracentrifugation. The long helical bundle-like topology of Munc 13-1 was first revealed by analysis of our data. In addition, these purified recombinant proteins provide ideal starting materials for further biochemical, biophysical, and structural studies on mammalian Munc 13 proteins.Munc13-1 is an essential component of synaptic vesicle releasing machinery. Three rat Munc 13-1 constructs were rationally designed based on homology and function, overexpressed in Escherichia coli, and purified to homogeneity with a final yield higher than 2 ~tg/ml of cell culture. The purified Munc 13-1 recombinant proteins had distinct oligomeric states, monodispersity and homogeneity properties. Their secondary structural contents were analyzed by the circular dichroism method, and the sedimentation coefficients of these recombinant proteins were measured by analytical ultracentrifugation. The long helical bundle-like topology of Munc 13-1 was first revealed by analysis of our data. In addition, these purified recombinant proteins provide ideal starting materials for further biochemical, biophysical, and structural studies on mammalian Munc 13 proteins.

关 键 词：Munc 13-1 MONODISPERSITY circular dichroism analytical ultracentrifugation sedimentationcoefficients 

分 类 号：Q632[生物学—生物物理学]