Separation and identification of hormone-active compounds using a combination of chromatographic separation and yeast-based reporter assay.

Arxula adeninivorans-based yeast cell assays for the detection of steroid hormones demonstrated their efficiency for the determination of total hormone activity in a variety of samples using a microtiter plate format. In this study, a preliminary chromatographic separation using thin-layer chromatography plates is introduced in order to allow a rapid identification of the compounds responsible for this hormonal activity. The yeast whole cell assay can then be performed on the plate, producing a detectable signal where a steroid hormone is present. Simultaneous detection of estrogens, progestogens and androgens on the same plate in the picogram range was achieved, while keeping the assay as simple and affordable as possible. The assay requires a single incubation of the thin-layer chromatography plate and the detection of reporter protein production can be performed by fluorescence scanning of the plate at different wavelengths. The chromatographic separation allows the separation of several estrogens, androgens and progestogens, thus making its application for 'real world' samples very useful. In this work, different water-based samples from environmental origins were used to demonstrate the capacity of this new bioassay. Trials showed that most samples, with the exception of complex samples such as wastewater influent, can be assayed.

Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor.

A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater.

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.

Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast.

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast (Arxula adeninivorans). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans, optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100ngml(-1). These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer.

Use of recombinant oestrogen binding protein for the electrochemical detection of oestrogen.

We have previously reported the development of an electrochemical method to quantitatively detect vertebrate oestrogens using an oestrogen binding protein (EBP1) present in wild type Saccharomyces cerevisiae and Candida albicans cells. However, the assays were complex and slow with both whole cells and cell lysate. In this work we report the transfer of the EBP1 gene to an industrial yeast, the addition of a his tag sequence to simplify purification of the protein, and the oestrogen binding characteristics of the protein. The recombinant protein (Ebp1p-6h) can now be produced by a non-pathogenic cell, and has shown good stability both when in use and when lyophilised. The detection range covers likely environmental concentrations of free oestrogens and the limit of detection is below the environmental concentration that has significant biological effect. In addition the assay period has been reduced to approximately 2min. This work reports progress toward the construction of a rapid, portable oestrogen sensor that is not restricted to use to the laboratory.