TY - CHAP
T1 - Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis
AU - Clémençon, Benjamin
AU - Fine, Michael
AU - Schneider, Philipp
AU - Hediger, Matthias A.
N1 - Funding Information:
This study was supported by the Swiss National Science Foundation (SNSF) TransCure and the TransCure International Fellowship Program (IFP TransCure), funded in part by an FP7 European Marie Curie Actions Grant.
Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - Detergents
KW - Hi-Clamp two-electrode voltage clamp system
KW - Human protein expression
KW - Membrane protein purification
KW - RoboInject system
KW - Single particle analysis and reconstruction
KW - Transmission electron microscopy
KW - Xenopus laevis oocytes
UR - http://www.scopus.com/inward/record.url?scp=84940001086&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84940001086&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2014.12.011
DO - 10.1016/bs.mie.2014.12.011
M3 - Chapter
C2 - 25857785
AN - SCOPUS:84940001086
T3 - Methods in Enzymology
SP - 241
EP - 265
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -