Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein

Autor: Patrick D Koks, Harry A. Kuiper, Richard J. R. Helsdingen, Ron L.A.P. Hoogenboom, Toine F.H. Bovee, Jaap Keijer
Jazyk: angličtina
Rok vydání: 2004
Předmět:
mice
receptor-alpha
Recombinant Fusion Proteins
Saccharomyces cerevisiae
Blotting
Western
Genetic Vectors
Green Fluorescent Proteins
RIKILT - Business Unit Veiligheid & Gezondheid
screen
s-cerevisiae
Sensitivity and Specificity
Green fluorescent protein
chemistry.chemical_compound
Transformation
Genetic
Genetics
Bioassay
Humans
Luciferase
Beta-galactosidase
cyc1 gene
Chlorophenol red
Luciferases
biology
Dose-Response Relationship
Drug
Estradiol
Estrogen Receptor alpha
Estrogens
General Medicine
assay
biology.organism_classification
beta-Galactosidase
Molecular biology
Yeast
disruption
Blotting
Southern
Luminescent Proteins
Biochemistry
chemistry
Gene Expression Regulation
Receptors
Estrogen
biology.protein
RIKILT - Business Unit Safety & Health
Biological Assay
activation
beta
transcription
Estrogen receptor alpha
Zdroj: Gene 325 (2004)
Gene, 325, 187-200
ISSN: 0378-1119
Popis: 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.
Databáze: OpenAIRE
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