Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.
We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.
Obourn
J D
JD
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Koszewski
N J
NJ
Notides
A C
AC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D011138:Polytetrafluoroethylene; D011960:Receptors, Estrogen / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013941:Thymine / Q000378:metabolism
D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol; D011138:Polytetrafluoroethylene; D004247:DNA; D005038:Ethylnitrosourea; D013941:Thymine