Abstract
Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E2) and wide dynamic range of more than four orders of magnitude.
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Acknowledgments
We thank Professor John A. Katzenellenbogen (University of Illinois, Urbana, IL, USA) for providing us the constructs pET15b-6xHisSRC-1 (NRBD) and pET15b-6xHis-ERα (LBD) [14]. We also thank Doctor Sari Mäkelä for providing us the compounds genistein, PPT and DPN. We thank Alun Parsons (FIMM) for reviewing the manuscript.
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Huovinen, T., Rytkönen, K., Lamminmäki, U. et al. A simple heterogeneous one-step assay for screening estrogenic compounds. Biotechnol Lett 35, 47–53 (2013). https://doi.org/10.1007/s10529-012-1050-0
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DOI: https://doi.org/10.1007/s10529-012-1050-0