Journal article
High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP
The Journal of steroid biochemistry and molecular biology, Vol.74(4), pp.169-178
2000
DOI: 10.1016/S0960-0760(00)00120-5
PMID: 11162922
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.
Details
- Title: Subtitle
- High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP
- Creators
- CHENG CHENG ZHANG - Department of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Avemte, Urbana, IL 61801, United StatesKevin A GLENN - Department of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Avemte, Urbana, IL 61801, United StatesMartin A KUNTZ - Department of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Avemte, Urbana, IL 61801, United StatesDavid J SHAPIRO - Department of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Avemte, Urbana, IL 61801, United States
- Resource Type
- Journal article
- Publication Details
- The Journal of steroid biochemistry and molecular biology, Vol.74(4), pp.169-178
- Publisher
- Elsevier Science
- DOI
- 10.1016/S0960-0760(00)00120-5
- PMID
- 11162922
- ISSN
- 0960-0760
- eISSN
- 1879-1220
- Language
- English
- Date published
- 2000
- Academic Unit
- General Internal Medicine; Internal Medicine
- Record Identifier
- 9984094651002771
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