tailieunhanh - Báo cáo khoa học: Cell-free expression as an emerging technique for the large scale production of integral membrane protein

Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expres-sion systems is in many cases the first major bottleneck. | ỊFEBS Journal MINIREVIEW Cell-free expression as an emerging technique for the large scale production of integral membrane protein Christian Klammt Daniel Schwarz Frank Lohr Birgit Schneider Volker Dotsch and Frank Bernhard Centre for Biomolecular Magnetic Resonance University of Frankfurt Main Institute for BiophysicalChemistry Frankfurt Main Germany Keywords cell-free expression G-protein coupled receptors integral membrane proteins NMR spectroscopy smallmultidrug transporter X-ray crystallography Correspondence F. Bernhard Centre for Biomolecular Magnetic Resonance Institute for BiophysicalChemistry University of Frankfurt Main Max-von-Laue-Str. 9 D-60438 Frankfurt Main Germany Fax 49 69 798 29632 Tel 49 69 798 29620 E-mail fbern@ Received 12 May 2006 revised 5 July 2006 accepted 11 July 2006 doi Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expression systems is in many cases the first major bottleneck. Preparative scale cell-free expression has now become an emerging alternative tool for the high level production of integral membrane proteins. Many toxic effects attributed to the overproduction of recombinant proteins are eliminated by cell-free expression as viable host cells are no longer required. A unique characteristic is the open nature of cell-free systems that offers a variety of options to manipulate the reaction conditions in order to protect or to stabilize the synthesized recombinant proteins. Detergents or lipids can easily be supplemented and membrane proteins can therefore be synthesized directly into a defined hydrophobic environment of choice that permits solubility and allows the functional folding of the proteins. Alternatively cell-free produced precipitates of membrane proteins can efficiently be solubilized in mild .

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