Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris.
The human mu-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).
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Centre National de la Recherche Scientifique, Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse Cedex 4, France. vsarra@ipbs.fr
Valérie
V
Sarramegna
Franck
F
Talmont
Marc
M
Seree de Roch
Alain
A
Milon
Pascal
P
Demange
D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D008164:Luminescent Proteins; D008565:Membrane Proteins; D017450:Receptors, Opioid, mu; D049452:Green Fluorescent Proteins