Novel method for evaluation of chemicals based on ligand-dependent recruitment of GFP labeled coactivator to estrogen receptor displayed on bacterial magnetic particles

摘要

We established a novel method to evaluate endocrine disrupting chemicals(EDCs)by assembling the estrogen receptor-ligand binding domain(ERLBD)and GFP labeled coactivator on magnetic nanoparticles.EDC can promote or inhibit coactivator recruitment to the ligand-ERLBD complex.ERLBD was displayed on the surface of nano-sized bacterial magnetic particles(BacMPs)produced by the magnetic bacterium,Magnetospirillum magneticum AMB-1.Our method resulted in 38 molecules of ERLBD molecules on a BacMPs with diameter of 75 nm.Furthermore,ligand-dependent recruitment assays of GFP labeled coactivator to ERLBD-BacMPs was performed by measuring the fluorescence intensity.17 beta-estradiol(E2),estriol,diethylstilbestrol,zeralenone(full agonist),octylphenol(partial agonist)and ICI 182,780(antagonist)were evaluated by this method.Full agonists tested showed increased fluorescence with increasing agonist concentration.Octylphenol had lower fluorescence intensity than E2.ICI 182,780 did not produce any fluorescence.The method developed in this study can evaluate the estrogenic potential of chemicals by discriminating whether they are an ER full agonist,partial agonist,or antagonist.Finally,this method is amenable adaptation into a high throughput format by using automated magnetic separation.