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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, Vol. 65, No. 2, 06.2009, p. 190-5.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Bleimling, N, Alexandrov, K, Goody, R & Itzen, A 2009, 'Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli', PROTEIN EXPRES PURIF, vol. 65, no. 2, pp. 190-5. https://doi.org/10.1016/j.pep.2008.12.002

APA

Bleimling, N., Alexandrov, K., Goody, R., & Itzen, A. (2009). Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli. PROTEIN EXPRES PURIF, 65(2), 190-5. https://doi.org/10.1016/j.pep.2008.12.002

Vancouver

Bleimling N, Alexandrov K, Goody R, Itzen A. Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli. PROTEIN EXPRES PURIF. 2009 Jun;65(2):190-5. https://doi.org/10.1016/j.pep.2008.12.002

Bibtex

@article{2d75ab33bd4e4d188fe1cb4cfdf8e309,
title = "Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli",
abstract = "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.",
keywords = "Chaperonin 10, Chaperonin 60, Enzyme Activation, Escherichia coli, Humans, Molecular Chaperones, Recombinant Fusion Proteins, rab GTP-Binding Proteins, Journal Article",
author = "Nathalie Bleimling and Kirill Alexandrov and Roger Goody and Aymelt Itzen",
year = "2009",
month = jun,
doi = "10.1016/j.pep.2008.12.002",
language = "English",
volume = "65",
pages = "190--5",
journal = "PROTEIN EXPRES PURIF",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

RIS

TY - JOUR

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 -