title: A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.
abstract: In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Jiangbi
authors.initials: J
authors.lastname: Xie
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Xiaocui
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Jinqiu
authors.initials: J
authors.lastname: Li
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Dongqin
authors.initials: D
authors.lastname: Zhao
authors.affiliation: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. Electronic address: mpzhao@pku.edu.cn.
authors.forename: Meiping
authors.initials: M
authors.lastname: Zhao
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D011994:Recombinant Proteins
keywords: 17β-Estradiol; Coumestrol; Estrogen receptor; Fluorescent assay; Homogeneous fluorescent assay; Neuron culture medium