The Purification Methods And Protein Property Studies On Vesicle Trafficking And Momentous Diseases Related Membrane Proteins

The process of vesicle translocation and fusion, as a ubiquitous and fundamental event in the cell growth and propagation, play a critical role in proteins trafficking and secretion. A lot of critical proteins are involved in multiple steps of the process, such as Munc13-1 and Exocyst complex. Munc13-1 is an essential component controlling the vesicle priming event, whereas the Exocyst complex involves in regulating the process of vesicle terthering and docking. Recent works on these proteins properties and characters, as well as the 3D structures provided useful information for learning their molecular mechanism of function. A series of Munc13-1 truncations were rational designed based on homology and function, overexpressed in E. coli system, and purified to homogeneity with chromatography. In addition, the highα-helix contents of these constructs were measured by circular dichroism method, and their distinct oligomeric states, monodispersity and homogeneity properties were analyzed and compared by gel chromatography and analytical ultracentrifugation. Furthermore, a topology structure of long helical-bundle like of Munc13-1 and a model that Munc13 acts as a monomer in vivo were first raised by analysis of our data.Through the trials of overexpression and purification of Sec10, one subunit of Exocyst complex, we concluded that Sec10 folds closely with its full-length amino acids because any truncation either in the amino (N)-terminal or in the carboxyl (C)-terminal induced error-folded structures, resulting in the formation of inclusion bodies. The highly polymerized Sec10 showed globular structure in solution by electron microscope.GABAB Receptor belongs to class C family of G-protein coupled receptors, its function disorder results in lots of nervous diseases, such as Alzheimer, Parkinsonismus, et al. We used hypothermal expression and inclusion body denaturation/renaturation methods to express and purify enough yields of exocellular fragments of GABAB R for protein crystallization.Glucose transporter 4 (GLUT4), as a hot spot protein in the research of type II diabetes, has a crucial role in whole-body glucose homeostasis. To data, there has little report on the method of large-scale extraction and purification of GLUT4. High yield of GLUT4 was extracted and purified from fat tissues by methods of homogenate, differential centrifugation and ammonium sulfate precipitation. The paper developed a method of effective purification of GLUT4 from fat tissue for further protein crystallization.It is obvious that the bottleneck of membrane structural biology is the extraction, purification and crystallization of the membrane proteins. Recent works on the fields of trafficking and disease related membrane proteins in this paper shed new light on the further proteins crystallization.