Development of a novel method for screening of estrogenic compounds using nano-sized bacterial magnetic particles displaying estrogen receptor
Development of a novel method for screening of estrogenic compounds using nano-sized bacterial magnetic particles displaying estrogen receptor
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DOI：
10.1016/j.aca.2004.10.074
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发表时间：
2005-03
影响因子：
6.2
通讯作者：
T. Yoshino;Fukuichi Kato;H. Takeyama;M. Nakai;Y. Yakabe;T. Matsunaga
中科院分区：
文献类型：
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作者：
T. Yoshino;Fukuichi Kato;H. Takeyama;M. Nakai;Y. Yakabe;T. Matsunaga
在本研究中，磁性细菌磁螺杆菌AMB-1成功地制备了表面具有人类雌激素受体配体结合结构域(ERLBD)的纳米细菌磁性颗粒(BMP)。此外，利用显示ERLBD的BMPs建立了雌激素类化合物的非同位素结合分析方法。利用BMP膜特异性蛋白MMS16作为锚定分子，将ERLBD定位于BMP表面。以碱性磷酸酶结合17β-雌二醇为示踪剂，用磁分离的方法从AMB-1转化子中简单地提取ERLBD-BMP复合体，并用于检测。该受体的解离常数为2.3 nm。通过碱性磷酸酶的酶促反应导致发光强度的降低来评价抑制曲线。这种受体结合分析的总体简单导致了一种可以很容易地适应高通量格式的方法。此外，该方法可以集成到使用磁分离的全自动配体筛选系统中。
In this study, nano-sized bacterial magnetic particles (BMPs) displaying human estrogen receptor ligand binding domain (ERLBD) on the surface was successfully produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Furthermore, a non-isotopic binding assay for estrogenic compounds using the BMPs displaying ERLBD was developed. A BMP membrane-specific protein, Mms16, was used as an anchor molecule to localize ERLBD on the surface of BMPs. ERLBD–BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants and used for the assay based on the competitive binding of alkaline phosphatase conjugated 17β-estradiol (ALP-E2) as a tracer. Dissociation constant of the receptor was 2.3nM. Inhibition curves were evaluated by the decrease in luminescence intensity resulting from the enzymatic reaction of alkaline phosphatase. The overall simplicity of this receptor binding assay results in a method that can be easily adapted to a high throughput format. Moreover, this method can be integrated into a fully-automated ligand screening system using magnetic separation.