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Neufahrn bei Freising, Germany

Chamas A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Giersberg M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Friedrich K.,Carl Gustav Carus Institute | Sonntag F.,Fraunhofer Institute for Material and Beam Technology | And 5 more authors.
Protein Expression and Purification | Year: 2015

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 100 ng ml-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. © 2014 Elsevier Inc. Source


Chamas A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Nieter A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Pham H.T.M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Pham H.T.M.,Vietnam Academy of Science and Technology | And 6 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

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. © 2015 Springer-Verlag Berlin Heidelberg Source


Henseleit A.,TU Dresden | Pohl C.,TU Dresden | Kaltenbach H.-M.,Quo Data GmbH | Hettwer K.,Quo Data GmbH | And 5 more authors.
Biosensors | Year: 2015

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step. © 2015 by the authors. Source


Pham H.T.M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Pham H.T.M.,Vietnam Academy of Science and Technology | Giersberg M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Gehrmann L.,Institute fur Energie und Umwelttechnik E.V. IUTA | And 8 more authors.
Sensors and Actuators, B: Chemical | Year: 2015

An Arxula adeninivorans based microbial biosensor has been developed for the determination of pharmaceuticals and chemicals such as omeprazole, lansoprazole, β-naphtoflavone and methylcholanthrene within 5 h using biochemical detection and 4 h and 10 min using amperometric detection. The biosensor consists of genetically modified A. adeninivorans G1212/YRC102-hAhR-hARNT-phyK (hAhR - human arylhydrocarbon receptor; hARNT - human arylhydrocarbon receptor nuclear translocator; inducible phyK - derived from Klebsiella sp. ASR1) as the biological component and coupled with either a biochemical or an amperometric detection method. The combination between hAh receptor gene, hARNT and the A. adeninivorans-derived glucoseamylase promoter of the reporter gene (GAA) containing specific cyp1A1-derived core sequence created a construct which enabled specific induction by pharmaceuticals. This offers a new cell-based biosensor for the pharmaceutical determination. The half maximum effective concentration (EC50) and the limit of detection (LoD) were found to be 236.13, 95.01 and 174.72, 83.65 μg/l for omeprazole and lansoprazole, respectively. These two pharmaceuticals are among the most widely used internationally. Additionally A. adeninivorans G1212/YRC102-hAhR-hARNT-phyK cells allow the measurement in raw wastewater, i.e. not concentrated, unpurified and untreated which will allow on-site operation in sewage treatment plants. © 2015 Elsevier B.V. All rights reserved. Source


Pham H.T.M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Pham H.T.M.,Vietnam Academy of Science and Technology | Chamas A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Nieter A.,Leibniz Institute of Plant Genetics and Crop Plant Research | And 9 more authors.
Sensors and Actuators, B: Chemical | Year: 2016

The two Arxula adeninivorans-based bioassays (A-YGS and A-YGFS) described here provide sensitive and reliable screening for glucocorticoids in aquatic environments. The biocomponents of A-YGS and A-YGFS were constructed to carry the human glucocorticoid receptor (hGRα) and the phytase gene (phyK, derived from Klebsiella sp. ASR1) or a fluorescence dsRED gene (derived from the Discosoma sp.), as reporter genes. The responses of A-YGS and A-YGFS were measured photometrically and spectrofluorometrically respectively. The half effective concentration (EC50) and limit of detection (LoD) values for dexamethasone obtained with A-YGS and A-YGFS were 0.81 and 0.29 μM, and 9.42 and 0.47 μM, respectively. Furthermore, both bioassays exhibited different binding specificities for several natural and synthetic glucocorticoids: A-YGS - corticosterone > cyproterone acetate > mifepristone > dexamethasone > cortisol > methylprednisolone > prednisolone and A-YGFS - cortisol >corticosterone >dexamethasone >prednisolone >betamethasone >methylprednisolone. As proof of principle, A-YGS was used to assay total glucocorticoids in river water, wastewater influent and effluent taken from a wastewater treatment plant in Germany. Glucocorticoids were not detected in both A-YGS and LC-MS/MS in seven of the eight samples, however a dexamethasone equivalent (DEQ) was detected in an influent wastewater sample using A-YGS (0.055 μM), while LC-MS/MS analysis showed that hydrocortisone and cortisone were present at 0.063 and 0.083 nM, respectively. © 2015 Elsevier B.V. All rights reserved. Source

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