title: Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater.
abstract: This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany.
authors: Alexandre
authors: A
authors: Chamas
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany.
authors: Annabel
authors: A
authors: Nieter
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany.|Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Vietnam.
authors: Ha Thi Minh
authors: HT
authors: Pham
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany.
authors: Martin
authors: M
authors: Giersberg
authors: QuoData GmbH, Prellerstr. 14, 01309, Dresden, Germany.
authors: Karina
authors: K
authors: Hettwer
authors: QuoData GmbH, Prellerstr. 14, 01309, Dresden, Germany.
authors: Steffen
authors: S
authors: Uhlig
authors: New diagnostics GmbH, Moosstr. 92c, 85356, Freising, Germany.
authors: Kirsten
authors: K
authors: Simon
authors: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, 8041, New Zealand.
authors: Keith
authors: K
authors: Baronian
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany. kunzeg@ipk-gatersleben.de.
authors: Gotthard
authors: G
authors: Kunze
mesh_terms: D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D057230:Limit of Detection; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011374:Progesterone / Q000032:analysis* / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic; D062065:Wastewater / Q000032:analysis*; D014874:Water Pollutants, Chemical / Q000032:analysis* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011374:Progesterone; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D062065:Wastewater; D014874:Water Pollutants, Chemical; C413663:fluorescent protein 583
keywords: Arxula adeninivorans; Progesterone; Reed-bed wastewater treatment plant; Transactivation assay