Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain

Coffer, A., Cavailles, V., Knowles, P., Pappin, D. (August 1996) Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain. J Steroid Biochem Mol Biol, 58 (5-6). pp. 467-77. ISSN 0960-0760 (Print)0960-0760 (Linking)

DOI: 10.1016/0960-0760(96)00068-4


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.

Item Type: Paper
Uncontrolled Keywords: Animals Binding Sites Escherichia coli/genetics/metabolism Estrogens/ metabolism Gene Transfer Techniques Mice Radioligand Assay Receptors, Estrogen/ analysis/genetics/metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein receptor
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Matt Covey
Date: August 1996
Date Deposited: 22 May 2014 15:48
Last Modified: 22 May 2014 15:48
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