BIOCHEMICAL-CHARACTERIZATION AND NOVEL ISOLATION OF PURE ESTROGEN-RECEPTOR HORMONE-BINDING DOMAIN

Biologically active, mouse estrogen receptor hormone-binding domain (r esidues 313-599) overexpressed in Escherichia coli was purified to app arent homogeneity as a single component with a molecular mass of 32.83 1 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The int act domain was isolated using a novel, rapid purification scheme witho ut recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity f or estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [H-3]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroi d unoccupied ERhbd (from dynamic light scattering) was similar to 72 k Da, suggesting that the pure, unlabelled ERhbd formed homodimers. Ster oid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [H-3]estradiol was unaffected by Ca2+ a nd Mg2+ ions up to 1 mM but was significantly inhibited by Zn2+ ions a t concentrations above 10 mu M, an effect reversed by EDTA. Copyright (C) 1996 Elsevier Science Ltd.