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Cell-free Expression (cell-free + expression)
Selected AbstractsEvaluation of detergents for the soluble expression of ,-helical and ,-barrel-type integral membrane proteins by a preparative scale individual cell-free expression systemFEBS JOURNAL, Issue 23 2005Christian Klammt Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation sttif. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial ,-helical multidrug transporter, EmrE, the ,-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy- sn -glycero-3-[phospho- rac -(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a ,-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. [source] Cell-free expression and selective isotope labelling in protein NMRMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2006David Staunton Abstract Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15N and 13C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell-free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1H15N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment. Copyright © 2006 John Wiley & Sons, Ltd. [source] Evaluation of detergents for the soluble expression of ,-helical and ,-barrel-type integral membrane proteins by a preparative scale individual cell-free expression systemFEBS JOURNAL, Issue 23 2005Christian Klammt Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation sttif. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial ,-helical multidrug transporter, EmrE, the ,-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy- sn -glycero-3-[phospho- rac -(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a ,-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. [source] Expression of functional Candida antarctica lipase B in a cell-free protein synthesis system derived from Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 2 2009Chang-Gil Park Abstract This article reports the cell-free expression of functional Lipase B from Candida antarctica (CalB) in an Escherichia coli extract. Although most of the cell-free synthesized CalB was insoluble under conventional reaction conditions, the combined use of molecular chaperones led to the soluble expression of CalB. In addition, the functional enzyme was generated by applying the optimal redox potential. When examined using p -nitrophenyl palmitate as a substrate, the specific activity of the cell-free synthesized CalB was higher than that of the reference protein produced in Pichia pastoris. These results highlight the potential of cell-free protein synthesis technology as a powerful platform for the rapid expression, screening and analysis of industrially important enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||