Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry.
Complementary DNA fragments that encode central and C-terminal domains of rainbow trout estrogen receptor (rtER) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). Both fusion proteins were induced by IPTG and could readily be detected as a 53-55 kDa band in crude extracts or in insoluble fraction after polyacrylamide gel electrophoresis and Coomassie blue staining. These recombinant proteins were solubilized and partially purified (ca. 60-75%) using centrifugation and different concentrations of urea. Gel mobility shift assays revealed that the hybrid protein containing ER central domain forms a specific complex with a synthetic estrogen-response-element. Similarly, we showed by steroid-binding assays that the hybrid protein containing the ER C-terminal domain binds specifically estrogen and not other steroids. These hybrid receptors were further isolated by electroelution after electrophoresis and used to immunize rabbits. Polyclonal antibodies from each antiserum were purified using GST-rtER fusion proteins. The specificity of these purified antibodies was confirmed by Western blot analysis using extracts from yeast and COS-1 cells transfected with rtER cDNA expression vectors. In these cells, rtER level was about 300-500 fmol/mg of protein, and the receptor was found as a single band migrating as a 65 kDa polypeptide. Interestingly, Western blot analysis with both purified antibodies directed against central or C-terminal regions of rtER revealed two receptor forms in trout liver nuclear extracts: a major form migrating as 65 kDa protein also observed in transfected cells, and a minor band at 71 kDa specific to the liver. Both receptor form levels were strongly induced by estradiol whereas they were virtually undetectable in untreated male trout livers. Immunocytochemistry performed on brain and pituitary of female trout revealed the presence of rtER in neurons located in the ventral telencephalon, preoptic area and mediobasal hypothalamus, as well as cells in the proximal pars distalis of the pituitary.
Pakdel
F
F
Laboratoire de Biologie Moléculaire, URA CNRS 256, Université de Rennes I, France.
Petit
F
F
Anglade
I
I
Kah
O
O
Delaunay
F
F
Bailhache
T
T
Valotaire
Y
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D001923:Brain Chemistry; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D004967:Estrogens / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D008297:Male; D017686:Oncorhynchus mykiss / Q000235:genetics*; D010902:Pituitary Gland / Q000737:chemistry / Q000648:ultrastructure; D011960:Receptors, Estrogen / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D000906:Antibodies; D004967:Estrogens; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D004247:DNA