Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies.
Cell-free expression techniques have emerged as promising tools for the production of membrane proteins for structural and functional analysis. Elimination of toxic effects and a variety of options to stabilize the synthesized proteins enable the synthesis of otherwise difficult to obtain proteins. Modifications in the reaction design result in preparative scale production rates of cell-free reactions and yield in milligram amounts of membrane proteins per one millilitre of reaction volume. A diverse selection of detergents can be supplied into the reaction system without inhibitory effects to the translation machinery. This offers the unique opportunity to produce a membrane protein directly into micelles of a detergent of choice. We present detailed protocols for the cell-free production of membrane proteins in different modes and we summarize the current knowledge of this technique. A special emphasize will be on the production of soluble and functionally folded membrane proteins in presence of suitable detergents. In addition, we will highlight the advantages of cell-free expression for the structural analysis of membrane proteins especially by liquid state nuclear magnetic resonance spectroscopy and we will discuss new strategies for structural approaches.
Schwarz
Daniel
D
Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany.
Klammt
Christian
C
Koglin
Alexander
A
Löhr
Frank
F
Schneider
Birgit
B
Dötsch
Volker
V
Bernhard
Frank
F
D001671:Biochemistry / Q000379:methods*; D002474:Cell-Free System / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D012995:Solubility
D003902:Detergents; D008565:Membrane Proteins