Abstract
In combining fluorescence measurements with ligand binding assays, the versatility of the EGFP C-terminally fused to the human mu opioid receptor (EGFP-hMOR) has been exploited to notably improve the expression level of functional G protein-coupled receptors in Drosophila S2 cells. A selected array of efficient optimization approaches is presented herein, ranging from a cell-sorting method, allowing for a substantial enrichment in EGFP-hMOR expressing cells, to the addition of chemical and pharmacological chaperones, significantly enhancing the yield and the activity of the expressed receptors. Consistent with previous studies, significant discrepancies were observed between the total amounts of fluorescent receptors over a limited subpopulation capable of ligand binding, even after expression optimization. Subsequently, membrane isopycnic centrifugation experiments allowed to separate the ligand binding active from the non-active membrane fraction, the latter most probably containing misfolded receptors. Taken together, these results illustrate a coherent set of advantageous productive and preparative methods for the production of GPCRs in the highly valuable Drosophila S2 expression system.
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Abbreviations
- DMSO:
-
Dimethyl sulfoxide
- EGFP:
-
Enhanced green fluorescent protein
- FBS:
-
Fetal bovine serum
- GPCR:
-
G protein-coupled receptor
- hMOR:
-
Human mu opioid receptor
- PBS:
-
Phosphate-buffered saline
- S2 cells:
-
Drosophila Schneider 2 cells
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Acknowledgements
We are very grateful to R. Benmaamar for helpful comments and critical reading of the manuscript. This work was supported by the Centre National pour la Recherche Scientifique. B.P. and V.K. were recipient of grants from the French Ministère de l’Education Nationale, de la Recherche et des Technologies.
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Karl Brillet and Bénédicte G. Perret contributed equally to this paper.
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Brillet, K., Perret, B.G., Klein, V. et al. Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells. Cytotechnology 57, 101–109 (2008). https://doi.org/10.1007/s10616-008-9125-4
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DOI: https://doi.org/10.1007/s10616-008-9125-4