Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli.
The guanine nucleotide binding protein Rab8A controls the final steps of exocytosis in mammalian cells. It has been implicated in the regulation of apical protein localization in intestinal epithelial cells and ciliary biogenesis. The in vitro structural and biochemical characterization of Rab8A and its interaction with regulator and effector molecules has been hampered by its insolubility in Escherichia coli expression systems. The conventional refolding procedure is laborious and yields only minute amounts of C-terminally truncated Rab8A (Rab8A(1-183): amino acids 1-183), not the full-length protein. Here, we report a method of expressing soluble, hexahistidine-tagged full-length human Rab8A from E. coli. The Rab8A gene was codon-optimized and coexpressed with bacterial GroEL and GroES chaperones. After two-step purification by Ni(2+) affinity chromatography and gel filtration, Rab8A was obtained at a yield of 4 mg protein per 1L of bacterial cell culture and a purity of >95%. The resultant protein was functionally active, as determined by GTPase activity and its interaction with the nucleotide exchange factor MSS4.
Bleimling
Nathalie
N
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Alexandrov
Kirill
K
Goody
Roger
R
Itzen
Aymelt
A
D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018835:Chaperonin 10; D018834:Chaperonin 60; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins