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Methods Enzymol. 2015;556:241-65. doi: 10.1016/bs.mie.2014.12.011. Epub 2015 Mar 20.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Author information

1
Institute of Biochemistry and Molecular Medicine (IBMM), University of Bern, Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland. Electronic address: benjamin.clemencon@ibmm.unibe.ch.
2
Institute of Biochemistry and Molecular Medicine (IBMM), University of Bern, Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.

Abstract

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

KEYWORDS:

Detergents; Hi-Clamp two-electrode voltage clamp system; Human protein expression; Membrane protein purification; RoboInject system; Single particle analysis and reconstruction; Transmission electron microscopy; Xenopus laevis oocytes

PMID:
25857785
DOI:
10.1016/bs.mie.2014.12.011
[Indexed for MEDLINE]

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