Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli
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Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli. / Bleimling, Nathalie; Alexandrov, Kirill; Goody, Roger; Itzen, Aymelt.
in: PROTEIN EXPRES PURIF, Jahrgang 65, Nr. 2, 06.2009, S. 190-5.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli
AU - Bleimling, Nathalie
AU - Alexandrov, Kirill
AU - Goody, Roger
AU - Itzen, Aymelt
PY - 2009/6
Y1 - 2009/6
N2 - 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.
AB - 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.
KW - Chaperonin 10
KW - Chaperonin 60
KW - Enzyme Activation
KW - Escherichia coli
KW - Humans
KW - Molecular Chaperones
KW - Recombinant Fusion Proteins
KW - rab GTP-Binding Proteins
KW - Journal Article
U2 - 10.1016/j.pep.2008.12.002
DO - 10.1016/j.pep.2008.12.002
M3 - SCORING: Journal article
C2 - 19116169
VL - 65
SP - 190
EP - 195
JO - PROTEIN EXPRES PURIF
JF - PROTEIN EXPRES PURIF
SN - 1046-5928
IS - 2
ER -