Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli.
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.
Wittliff
J L
JL
Department of Biochemistry, University of Louisville, Kentucky 40292.
Wenz
L L
LL
Dong
J
J
Nawaz
Z
Z
Butt
T R
TR
D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D004958:Estradiol / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics
D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D004958:Estradiol