Abstract
The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the lambda PL promoter in Escherichia coli. Analysis of extracts by Western blot showed that intact receptor protein was produced only when PL promoter was depressed by nalidixic acid at 30 degrees C. To ascertain the intactness of the expressed protein, estrogen receptor in bacterial extracts was titrated with either 17 beta-[3H]estradiol or 17 beta-[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 x 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 immuno-recognition 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.
Highlights
19406-0939 and the James Graham Brown Cancer of Pharmacology, University of Louisville, SmithKline Beecham Pharmaceuticals, Center, The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the X 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
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
Summary
T o facilitate th e expression of full length human estrogen receptor in E. coli,the carboxyl-termi na l glycine oCthe ublqu ltin gene was fused to the amino terminus of the receptor gene. To evaluate its integrity further, the ligand binding specificity of E. coli-expressed ER was compared simultaneously with a preparation of wild-type ER exhibiting a binding affinity (Kd value) similar to the bacterial extract employed (Fig. 4). Various compounds such as estradiol, diethylstilbestrol, and the antiestrogenic therapeutic drugs, tamoxifen and toremifene, are known to inhibit estrogen binding by wild-type ER (e.g. Ref. 26). Increasing concentrations of nonradioactive ERE in the reaction mixture effectively competed with the labeled ERE (lanes 4-7) In this experiment, the DNA probe was labeled with polynucleotide kinase and [32PJATP; as a result of endogenous alkaline phosphatases present in E. coli extract, some cleavage of the terminal 32p was observed (Fig. 7). These studies demonstrate that E. coliexpressed ubiquitin-ER retains its specific ERE binding properties
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