Application of Oestrogen Receptor Ligand Binding Domain to the Generic Isolation of Oestrogens by Receptor Affinity Chromatography

Abstract

Human oestrogen receptor (alpha) ligand binding domain (hER-LBD) was expressed in E. coli and isolated using a novel approach. The solubilised recombinant receptor had the expected biological activity in terms of ligand binding affinity and selectivity, indicating the potential for use in the proposed receptor affinity chromatography (RAC) application. Subsequent covalent binding of hER-LBD to agarose support provided an affinity matrix capable of selective binding of oestrogenic ligands, with a capacity for 17β-oestradiol of ∼6 ng/mL wet gel. In initial studies, a yield of ∼75% of bound ligand from the affinity matrix was obtained by elution with aqueous ethanol. Immobilised hER-LBD eluted with ethanol retained the majority of its capability to bind 17β-oestradiol (E2), indicating the possibility of reuse of the receptor matrix. In ligand-receptor displacement studies, using [³H]E2-saturated immobilised hER-LBD, direct extraction of the xenoestrogen 2′,3′,4′,5′-tetrachloro-4-biphenylol (TeCBol) from a model food (aqueous gelatin solution) was inhibited at the highest concentration of gelatin tested (1%), however, prior precipitation and extraction with ethanol enabled dose dependent binding of TeCBol. The present studies thus provide preliminary proof of principle for the application of hER-LBD for the purpose of RAC and for the generic extraction of oestrogens and xenoestrogens from biological matrices.