Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells
Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells
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DOI：
10.1007/s10616-008-9125-4
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发表时间：
2008-05-01
期刊：
影响因子：
2.2
通讯作者：
Wagner, Renaud
中科院分区：
文献类型：
--
作者：
Brillet, Karl;Perret, Benedicte G.;Wagner, Renaud
将荧光测量与配体结合测定相结合，利用与人 mu 阿片受体 (EGFP-hMOR) 融合的 EGFP C 端的多功能性来显着提高果蝇 S2 细胞中功能性 G 蛋白偶联受体的表达水平。本文提出了一系列选定的有效优化方法，从允许大量富集 EGFP-hMOR 表达细胞的细胞分选方法，到添加化学和药理学伴侣，显着提高表达受体的产量和活性。与之前的研究一致，即使在表达优化之后，在能够结合配体的有限亚群中，荧光受体的总量之间也观察到显着差异。随后，膜等密度离心实验允许将配体结合活性部分与非活性膜部分分离，后者很可能含有错误折叠的受体。总而言之，这些结果说明了在高价值的果蝇 S2 表达系统中生产 GPCR 的一套连贯的有利生产和制备方法。
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.