Membrane protein expression in the eyes of transgenic flies.
Eukaryotic membrane proteins are often difficult to obtain in sufficient amounts for structural studies using classical cell culture overexpression systems. This could be due to incomplete protein maturation and insufficient trafficking to the plasma membrane or to a cytotoxic effect of the recombinant membrane protein changing the overall metabolism. The method presented here takes advantage of the membrane stacks in the retina, where rhodopsin resides. The idea is to direct G protein-coupled receptors (GPCRs) and transporters to these membrane stacks and to express the target proteins in the retina of transgenic Drosophila melanogaster. Drosophila was chosen since fly genetics are well established and rather easily accessible. Metabotropic glutamate receptor mGluRa from Drosophila was among the first examples for GPCRs expressed in this system. It showed high expression yield, functionality and high homogeneity. When the same protein was expressed in Sf9 cells, however, contamination by immature protein was a problem. Encouraged by this success, the fly system is now successfully used for a larger variety of membrane proteins, including mammalian GPCRs and transporters.
Panneels
Valérie
V
Heidelberg University Biochemistry Center, Germany.
Sinning
Irmgard
I
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004331:Drosophila melanogaster / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D012160:Retina / Q000166:cytology*; D014162:Transfection; D015854:Up-Regulation
D026901:Membrane Transport Proteins; D043562:Receptors, G-Protein-Coupled