title: High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.
abstract: 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.
authors.affiliation: Department of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Zhang
authors.forename: K A
authors.initials: KA
authors.lastname: Glenn
authors.forename: M A
authors.initials: MA
authors.lastname: Kuntz
authors.forename: D J
authors.initials: DJ
authors.lastname: Shapiro
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000502:physiology; D014475:Uncoupling Agents / Q000494:pharmacology*
chemical_list: D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D014475:Uncoupling Agents; D004958:Estradiol; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004247:DNA