Résumé
Biologically active, mouse estrogen receptor hormone-binding domain (residues 313–599) overexpressed in
Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [
3H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was ∼ 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [
3H]estradiol was unaffected by Ca
2+ and Mg
2+ ions up to 1 mM but was significantly inhibited by Zn
2+ ions at concentrations above 10 μM, an effect reversed by EDTA.