High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

Dimerizing the estrogen receptor DNA binding domain enhances binding to estrogen response elements.

In this work, we provide a rationale for the finding that the estrogen receptor (ER) binds to its DNA response element as a homodimer in vivo. Binding of the monomer estrogen receptor DNA binding domain (ER DBD) to a palindromic, consensus estrogen response element (ERE) is increased 5-6-fold when the ER DBD is dimerized either by a monoclonal antibody that recognizes an attached epitope tag or by expressing the ER DBD as a single molecule in which the two monomers are joined by a peptide linker. Most of the increase in binding is due to stabilization of the ER DBD.ERE complex. We observed only an approximately 2.5-fold reduction in binding when a consensus ERE was replaced with widely spaced ERE half-sites, suggesting that the interaction between ER DBDs on the ERE is relatively weak, and that in full-length ER the DBDs can move independently of each other. To test binding to an imperfect palindrome, typical of the imperfect EREs found in almost all natural estrogen receptor responsive genes, we used the pS2 ERE. Even at high concentrations of ER DBD, specific binding of the ER DBD to the imperfect pS2 ERE was undetectable. Both of the dimerized ER DBDs exhibited efficient binding to the imperfect pS2 ERE, with an affinity at least 25-fold greater than monomer ER DBD. These data support the view that steroid receptor dimerization provides an important mechanism facilitating the recognition of naturally occurring, imperfect hormone response elements.