Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris
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- 作者：Sarramegna ; Valé ; rie ; Talmont ; Franck ; Seree de Roch ; Marc ; Milon ; Alain ; Demange ; Pascal
- 关键词：Membrane protein ; GPCR ; Pichia pastoris ; GFP ; Heterologous expression ; HuMOR ; human μ ; -opioid receptor ; AOX1 ; alcohol oxidase-1 ; α ; -MF ; alpha mating factor ; GFP ; green fluorescent protein ; BMGY ; buffered minimal glycerol-complex medium ; BMMY ; buff
- 刊名：Journal of Biotechnology
- 出版年：2002
- 出版时间：October 9, 2002
- 年：2002
- 卷：99
- 期：1
- 页码：23-39
- 全文大小：404 K
文摘
The human μ-opioid receptor (HuMOR) was fused in its N-terminus end to the green fluorescent protein (GFP) or/and to the c-myc and six histidines tags in its C-terminus end, and expressed in the methylotrophic yeast Pichia pastoris. Neither the C- nor the N-terminal tagging of the receptor does modify its pharmacological properties as compared to the untagged receptor. Expression levels of fusion receptors determined by GFP fluorescence measurements strongly correlates with the number of sites expressed per cell detected through saturation studies (Bmax value), thus showing that GFP is an efficient and reliable reporter of the HuMOR functional expression. The N- and C-terminus tags have allowed to show that the entire molecule is overexpressed. They have permitted in-situ localization experiments using fluorescence and electron microscopy techniques and have shown a dense intracellular labelling. Above all, the quantification of expression levels made possible through fluorescence intensity analysis, have revealed that huge amounts of receptor are produced that could not be detected through classical binding experiments: for a Bmax value of 1 pmol mg−1 of receptor determined through binding studies, 16 pmol were found in membrane preparations using fluorescence and 100 pmol in whole cells. These results should be very useful for large-scale production and structural biology of HuMOR, and other G-protein coupled receptors (GPCRs).