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 Escherich ia 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 poly acrylamide gel electrophoresis and Coomassie blue staining. These reco mbinant proteins were solubilized and partially purified (ca. 60-75%) using centrifugation and different concentrations of urea. Gel mobilit y shift assays revealed that the hybrid protein containing ER central domain forms a specific complex with a synthetic estrogen-response-ele ment. Similarly, we showed by steroid-binding assays that the hybrid p rotein 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 con firmed by Western blot analysis using extracts from yeast and COS-1 ce lls transfected with rtER cDNA expression vectors. In these cells, rtE R level was about 300-500 fmol/mg of protein, and the receptor was fou nd as a single band migrating as a 65 kDa polypeptide. Interestingly, Western blot analysis with both purified antibodies directed against c entral or C-terminal regions of rtER revealed two receptor forms in tr out liver nuclear extracts: a major form migrating as 65 kDa protein a lso observed in transfected cells, and a minor band at 71 kDa specific to the liver. Both receptor form levels were strongly induced by estr adiol whereas they were virtually undetectable in untreated male trout livers. Immunocytochemistry performed on brain and pituitary of femal e trout revealed the presence of rtER in neurons located in the ventra l telencephalon, preoptic area and mediobasal hypothalamus, as well as cells in the proximal pars distalis of the pituitary.