Hossain Shakhawat, Hossain Md Forhad, Sohan Md Sohanur Rahman, Omori Yuki, Amin Mohammad Tohidul, Tokumoto Toshinobu
Department of Bioscience, Graduate School of Science and Technology, National University Corporation Shizuoka University, Shizuoka, Japan.
PLoS One. 2025 Sep 19;20(9):e0332765. doi: 10.1371/journal.pone.0332765. eCollection 2025.
G protein-coupled estrogen receptor (GPER) is a member of the GPCR family and a key mediator of the rapid, nongenomic actions of estrogens as membrane estrogen receptors. In this study, we established a procedure for the expression and purification of recombinant human membrane estrogen receptor (hGPER) protein via the expression system using the methylotrophic yeast Pichia pastoris. By optimizing codon usage, we successfully expressed hGPER at a level that can be purified by column chromatography. The recombinant protein was purified via three chromatography steps. Purified hGPER showed specific estrogen-binding activity (Kd = 9.9 nM and Bmax = 1.76 nM) in a radiolabeled steroid-binding assay. We subsequently established a homogeneous assay for hGPER ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to hGPER. GQDs coupled with hGPER (GQD-hGPER) caused a decrease in fluorescence at 520 nm from E2-BSA-FITC, which was activated by 370 nm light upon the addition of free estradiol to the reaction mixture. Fluorescence was decreased by the administration of hGPER ligands but not by steroids that do not interact with hGPER. Thus, we successfully established a ligand-binding assay for hGPER that is suitable for screening potential compounds. hGPER is a promising candidate for drug discovery for nongenomic estrogen-stimulating effects. The homogeneous assay established in this study will be usable for that purpose.
G蛋白偶联雌激素受体（GPER）是GPCR家族的成员，作为膜雌激素受体，是雌激素快速非基因组作用的关键介质。在本研究中，我们通过利用甲基营养型酵母毕赤酵母表达系统，建立了一种重组人膜雌激素受体（hGPER）蛋白的表达和纯化程序。通过优化密码子使用，我们成功表达了可通过柱色谱纯化的hGPER。重组蛋白通过三步色谱法进行纯化。在放射性标记类固醇结合试验中，纯化的hGPER表现出特异性雌激素结合活性（Kd = 9.9 nM，Bmax = 1.76 nM）。随后，我们通过将称为石墨烯量子点（GQDs）的半导体纳米颗粒与hGPER偶联，建立了hGPER配体的均相测定法。与hGPER偶联的GQDs（GQD-hGPER）导致E2-BSA-FITC在520 nm处的荧光降低，在反应混合物中加入游离雌二醇后，该荧光在370 nm光下被激活。hGPER配体的给药会降低荧光，但与hGPER不相互作用的类固醇则不会。因此，我们成功建立了一种适用于筛选潜在化合物的hGPER配体结合测定法。hGPER是用于非基因组雌激素刺激作用药物发现的有前景的候选物。本研究中建立的均相测定法将可用于该目的。
