title: Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain.
abstract: 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 approximately 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 Ca2+ and Mg2+ ions up to 1 mM but was significantly inhibited by Zn2+ ions at concentrations above 10 microM, an effect reversed by EDTA.
authors.affiliation: Protein Isolation and Cloning Laboratory, The Imperial Cancer Research Fund, London, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Coffer
authors.forename: V
authors.initials: V
authors.lastname: Cavailles
authors.forename: P
authors.initials: P
authors.lastname: Knowles
authors.forename: D
authors.initials: D
authors.lastname: Pappin
mesh_terms: D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D018014:Gene Transfer Techniques; D051379:Mice; D011869:Radioligand Assay; D011960:Receptors, Estrogen / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D004967:Estrogens; D011960:Receptors, Estrogen