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

Chengcheng Zhang, Kevin A. Glenn, Martin A. Kuntz, David J. Shapiro

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Abstract

The expression of high levels of full-length human estrogen receptor α (hERα) 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 hERα (fER) was expressed in E. coli at ∼ 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 17β-estradiol with the same affinity as hER expressed in human cells (KD ∼ 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.

Original languageEnglish (US)
Pages (from-to)169-178
Number of pages10
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume74
Issue number4
DOIs
Publication statusPublished - Nov 15 2000

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Keywords

  • Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)
  • Escherichia coli
  • Human estrogen receptor α (hERα)

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

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