increased penetration since the average distance that photons penetrate a specific material is determined by the photon energy, the type of material, and its density

ltures and their requirement to work sterile. The costs for making a transgenic fly and maintaining even large scale cultures is negligible. In addition, making a fly can be faster than producing baculovirus stocks for overexpression in insect cells. Due to the short life cycle of the flies, about one month is sufficient starting from the DNA-construct of the target MP to the first expression test with the transgenic fly. While an overall comparison of different expression systems is straightforward concerning the costs, the comparison of yields, workload and most importantly the protein quality requires more attention. Compared with expression systems that require liters of sterile Vercirnon supplier medium, the continuous fly cultures and the handling of small volumes provide important advantages. When the workload of membrane preparation and the quality of the purified MPs are compared with conventional expression systems, the fly eye system is superior. Taken together, we developed a fly eye system for the heterologous and homologous expression of different classes of eukaryotic membrane proteins. It offers a number 15963531 of advantages compared to conventional expression systems and is more easily accessible than one would probably imagine. The fly eye system opens the door for studying eukaryotic membrane proteins that have so 23472002 far not been accessible to biochemical and biophysical studies. Materials and Methods Cloning strategy MP targets: the rat mGluR5, human sodiumdependent serotonin transporter, glutamate transporters and channelrhodopsin constructs were generous gifts from J.-P. Pin, R. D. Blakely, S. Birman and P. Hegemann, respectively. The Drosophila melanogaster SERT cDNA from the Berkeley Drosophila Genome Project was provided by BioCat/Open Biosystems. The general protocol for cloning of target MPs has been described previously. Typically, the gene coding for the target MP was amplified using EcoRI and a XhoI restriction sites and cloned in frame with eGFP into the Drosophila pUAST April 2011 | Volume 6 | Issue 4 | e18478 Eukaryotic Membrane Protein Expression vector. GFP was flanked at the N-terminus by a Leu-Glu linker encoded by the XhoI site followed by the TEV-cleavage site ENLYFQG and at the C-terminus by a 6-his tag. The construct in pUAST was sequenced and tested for expression in Schneider S2 cells as described. #6-2380 ) pre-cooled in liquid nitrogen. After shaking, the heads were collected from the middle compartment and stored at 280uC. Membrane preparation Frozen fly heads were homogenized in sucrose buffer and membranes were prepared as described. It is noteworthy that fly eye tissue is much easier to homogenize than cells in culture. Transgenic fly generation The MP-GFP construct cloned in the pUAST vector was used for classical P-element-mediated transformation of embryos of the Drosophila host line w1118 or BestGene ). Most of the driver lines were provided by the Bloomington center. The various driver lines used in this study were eye-specific using either the minimal rhodopsin promoter for the Rh1-GAL4 line or a glass-binding enhancer element GMR for the GMR-GAL4 lines. The GMR driver lines used a pentameric arrangement of an enhancer region of the Rh1 promoter. The GMR8506 driver has a longer pentameric repeat than the GMR1104 driver . An advantage of the GMR8506 driver is that the longer enhancer site sequence confers a strict PRC specificity. The ELAV-GAL4 driver was chosen for its predominant induction of expression in neultures and their requirement to work sterile. The costs for making a transgenic fly and maintaining even large scale cultures is negligible. In addition, making a fly can be faster than producing baculovirus stocks for overexpression in insect cells. Due to the short life cycle of the flies, about one month is sufficient starting from the DNA-construct of the target MP to the first expression test with the transgenic fly. While an overall comparison of different expression systems is straightforward concerning the costs, the comparison of yields, workload and most importantly the protein quality requires more attention. Compared with expression systems that require liters of sterile medium, the continuous fly cultures and the handling of small volumes provide important advantages. When the workload of membrane preparation and the quality of the purified MPs are compared with conventional expression systems, the fly eye system is superior. Taken together, we developed a fly eye system for the heterologous and homologous expression of different classes of eukaryotic membrane proteins. It offers a number of advantages compared to conventional expression systems and is more easily accessible than one would probably imagine. The fly eye system opens the door for studying eukaryotic membrane proteins that have so far not been accessible to biochemical and biophysical studies. Materials and Methods Cloning strategy MP targets: the rat mGluR5, human sodiumdependent serotonin transporter, glutamate transporters and channelrhodopsin constructs were generous gifts from J.-P. Pin, R. D. Blakely, S. Birman and P. Hegemann, respectively. The Drosophila melanogaster SERT cDNA 10555746 from the Berkeley Drosophila Genome Project was provided by BioCat/Open Biosystems. The general protocol for cloning of target MPs has been described previously. Typically, the gene coding for the target MP was amplified using EcoRI and a XhoI restriction sites and cloned in frame with eGFP into the Drosophila pUAST April 2011 | Volume 6 | Issue 4 | e18478 Eukaryotic Membrane Protein Expression vector. GFP was flanked at the N-terminus by a Leu-Glu linker encoded by the XhoI site followed by the TEV-cleavage site ENLYFQG and at the C-terminus by a 6-his tag. The construct in pUAST was sequenced and tested for expression in Schneider S2 cells as described. #6-2380 ) pre-cooled in liquid nitrogen. After shaking, the heads were collected from the middle compartment and stored at 280uC. Membrane preparation Frozen fly heads were homogenized in sucrose buffer and membranes were prepared as described. It is noteworthy that fly eye tissue is much easier to homogenize than cells in culture. Transgenic fly generation The MP-GFP construct cloned in the pUAST vector was used for classical P-element-mediated transformation of embryos of the Drosophila host line w1118 or BestGene ). Most of the driver lines were provided by the Bloomington center. The various driver lines used in this study were eye-specific using either the minimal rhodopsin promoter for the Rh1-GAL4 line or a glass-binding enhancer element GMR for the GMR-GAL4 lines. The GMR driver lines used a pentameric arrangement of an enhancer region of the Rh1 promoter. The GMR8506 driver has a longer pentameric repeat than the GMR1104 driver . An advantage of the GMR8506 driver is that the longer enhancer site sequence confers a strict PRC specificity. The ELAV-GAL4 driver was chosen for its predominant induction of expression in neu

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