Expression, purification and characterization of critical domains of Munc13-1.
Munc13-1 is an essential component of synaptic vesicle releasing machinery. Three rat Munc13-1 constructs were rationally designed based on homology and function, overexpressed in Escherichia coli, and purified to homogeneity with a final yield higher than 2 microg/ml of cell culture. The purified Munc13-1 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 Munc13-1 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 Munc13 proteins.
Ma
Cong
C
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.
Hou
Hai
H
Tian
Wei
W
Xu
Tao
T
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014461:Ultracentrifugation
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C103912:Unc13a protein, rat