Thu, 21 Aug 2008 10:14:23 BST CiteULike: Odi Schäfer http://www.citeulike.org/user/Odi/author/Schäfer CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/Odi/article/887334 <i>Magn Reson Chem, Vol. 44 Spec No (July 2006)</i><br /><br />Despite major technical advance in methods used for structural investigations of proteins structure determination of membrane proteins still poses a significant challenge. Recently, the application of cell-free expression systems to membrane proteins has demonstrated that this technique can be used to produce quantities sufficient for structural investigations for many different membrane proteins. In particular for NMR spectroscopy, cell-free expression provides major advantages since it allows for amino acid type selective and even amino acid position specific labeling. In this mini-review we discuss the combination of cell-free membrane protein expression and liquid state NMR spectroscopy. Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins. A Koglin C Klammt N Trbovic D Schwarz B Schneider B Schäfer F Löhr F Bernhard V Dötsch doi:10.1002/mrc.1833 Magn Reson Chem, Vol. 44 Spec No (July 2006) 2006-10-06T14:11:59-00:00 2006 Magn Reson Chem 0749-1581 44 Spec No cell-free expression membrane protein http://www.citeulike.org/user/Odi/article/602802 <i>Eur J Biochem, Vol. 271, No. 3. (February 2004), pp. 568-580.</i><br /><br />We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed alpha-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins. High level cell-free expression and specific labeling of integral membrane proteins. C Klammt F Löhr B Schäfer W Haase V Dötsch H Rüterjans C Glaubitz F Bernhard Eur J Biochem, Vol. 271, No. 3. (February 2004), pp. 568-580. 2006-04-26T15:05:57-00:00 2004 Eur J Biochem 0014-2956 271 3 568 580 cell-free detergents expression membrane protein system translation