HORMONE-BINDING AND DNA-BINDING MECHANISMS OF THE RECOMBINANT HUMAN ESTROGEN-RECEPTOR

We have investigated the hormone- and DNA-binding mechanisms of the wi ld-type human estrogen receptor (hER) overproduced in insect cells usi ng a baculovirus expression system. The recombinant hER was indistingu ishable in size (67 kDa) and immunogenically from the native human est rogen receptor in MCF-7 breast carcinoma cells. The recombinant hER wa s purified to 70-80% homogeniety with a two-step procedure that includ ed ammonium sulfate precipitation and oligonucleotide affinity chromat ography using a unique Teflon affinity matrix. The recombinant hER bou nd estradiol with a positively cooperative mechanism. At hER concentra tions in excess of 13 nM the Hill cofficient reached a maximal value o f 1.6, whereas, at lower hER concentrations, the Hill cofficient appro ached 1.0, suggesting that the hER was dissociated to the monomeric sp ecies 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 t wo-fold symmetry of the hER binding to the ERE with each monomer of th e hER bound in the major groove of the DNA. These data indicate that t he recombinant hER is valuable to define the biochemical and structura l properties of the native estrogen receptor.