Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein.
The aim of this study was to develop an estrogen transcription activation assay that is sensitive, fast and easy to use in the routine screening of estrogen activity in complex matrices such as agricultural products. Recombinant yeast cells were constructed that express the human estrogen receptor alpha (ER alpha) and beta-Galactosidase (beta Gal), Luciferase (Luc) or yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein. Compared to other yeast assays, these new cells contain both the receptor construct as well as the reporter construct stably integrated in the genome with only one copy of the reporter construct. Dose-response curves for 17beta-estradiol (E2) obtained with the beta Gal assay were similar to those reported and the calculated EC(50) of 0.2 nM was even slightly better. However, 5 days of incubation were required before the chlorophenol red product could be measured. The Luc assay was as sensitive as the beta Gal assay and gave an EC(50) of 0.2 nM, but the signals were rather low and, although the assay can be performed within 1 day, the procedure is laborious and caused variability. The yEGFP revealed an EC(50) of 0.4 nM, but compared to the beta Gal and the Luc assay, the response was much better. This yEGFP assay can be performed completely in 96 well plates within 4 h and does not need cell wall disruption nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. These qualities make that this yEGFP assay is suited to be used as a high throughput system.
Bovee
Toine F H
TF
RIKILT, Institute of Food Safety, 6700 AE Wageningen, The Netherlands. Toine.Bovee@wur.nl
Helsdingen
Richard J R
RJ
Koks
Patrick D
PD
Kuiper
Harry A
HA
Hoogenboom
Ron L A P
RL
Keijer
Jaap
J
D001681:Biological Assay / Q000379:methods*; D015139:Blotting, Southern; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D047628:Estrogen Receptor alpha; D004967:Estrogens; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004958:Estradiol; D008156:Luciferases; D001616:beta-Galactosidase