Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli

James L. Wittliff, Laura L. Wenz, Jing Dong, Zafar Nawaz, Tauseef R. Butt

Research output: Contribution to journalArticle

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Abstract

The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the λ PL promoter in Escherichia coli. Analysis of extracts by Western blot showed that intact receptor protein was produced only when PL promoter was derepressed by nalidixic acid at 30°C. To ascertain the intactness of the expressed protein, estrogen receptor in bacterial extracts was titrated with either 17β-[3H]estradiol or 17β-[125I]iodoestradiol, and the mass was quantified by enzyme immunoassay. Cell extracts contained 484 fmol of estrogen receptor per mg of soluble protein by titration with a Kd of 3.2 × 10-10 M. The simultaneous analysis by enzyme immunoassay resulted in 487 fmol/mg of extract protein indicating that most of the expressed protein bound ligand with wild type properties. Ligand competition analysis demonstrated that expressed receptor contained a binding domain preferentially recognizing estrogenic substances identical with that of wild-type estrogen receptor. The E. coli-expressed estrogen receptor also associated specifically with estrogen response DNA elements. Full length receptor protein was detected by ligand binding and immunorecognition using size exclusion chromatography. Hydrophobic interaction chromatography showed a major isoform which exhibited both ligand binding and immunorecognition identical with the estrogen receptor from human breast tumor tissues. These data indicate that estrogen receptor expressed in E. coli retained characteristics of the native protein found in human tissues.

Original languageEnglish
Pages (from-to)22016-22022
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number35
StatePublished - Dec 15 1990
Externally publishedYes

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Escherichia coli Proteins
Ubiquitin
Estrogen Receptors
Fusion reactions
Escherichia coli
Ligands
Proteins
Immunoenzyme Techniques
Tissue
Nalidixic Acid
Size exclusion chromatography
Response Elements
Enzymes
Chromatography
Cell Extracts
Titration
Hydrophobic and Hydrophilic Interactions
Gel Chromatography
Tumors
Estradiol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli. / Wittliff, James L.; Wenz, Laura L.; Dong, Jing; Nawaz, Zafar; Butt, Tauseef R.

In: Journal of Biological Chemistry, Vol. 265, No. 35, 15.12.1990, p. 22016-22022.

Research output: Contribution to journalArticle

Wittliff, James L. ; Wenz, Laura L. ; Dong, Jing ; Nawaz, Zafar ; Butt, Tauseef R. / Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 35. pp. 22016-22022.
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