Cloning, overexpression, purification and preliminary characterization of human septin 8.
Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T (m) of 54 degrees C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.
Souza
T A C B
TA
Institute of Biology, State University of Campinas, Campinas, SP, Brazil.
Barbosa
J A R G
JA
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058112:Septins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013696:Temperature
D003598:Cytoskeletal Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; C464445:SEPTIN8 protein, human; D058112:Septins