Development of a bioluminescence resonance energy-transfer assay for estrogen-like compound in vivo monitoring.
A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen alphareceptor (ERalpha) homodimerization as a result of estrogen-like compound binding. ERalpha monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERalpha dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERalpha-Rluc and ERalpha-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-beta estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-beta estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.
Michelini
Elisa
E
Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy.
Mirasoli
Mara
M
Karp
Matti
M
Virta
Marko
M
Roda
Aldo
A
D001426:Bacterial Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000032:analysis / Q000737:chemistry; D047628:Estrogen Receptor alpha / Q000737:chemistry*; D004967:Estrogens / Q000032:analysis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D049410:Luciferases, Renilla / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D004958:Estradiol; D049410:Luciferases, Renilla