RAINBOW-TROUT GLUCOCORTICOID RECEPTOR OVEREXPRESSION IN ESCHERICHIA-COLI - PRODUCTION OF ANTIBODIES FOR WESTERN BLOTTING AND IMMUNOHISTOCHEMISTRY

Fragments of cDNA that encode the N-terminal and DNA-binding domains ( DBD) of the rainbow trout gluco-corticoid receptor (rtGR) were express ed in Escherichia coli as fusion proteins with glutathione-S-transfera se (GST). The fusion proteins induced by IPTG could readily be detecte d as 45- and 40-kDa bands, respectively, in crude extracts, as well as in proteins purified on glutathione-agarose. These purified hybrid pr oteins were used to immunize rabbits. The antisera produced were teste d for specificity by Western blot analysis using extracts from COS-1 c ells transfected with an rtGR expression vector and from trout liver c ells. The antisera raised against the DBD domain did not detect any ba nds on Western blots, even at low antiserum dilution. However, the pur ified DBD fusion protein specifically bound GRE-containing DNA fragmen ts in gel-shift assays, and the retarded complexes were supershifted b y these antibodies. The antisera raised against the N-terminal domain consistently detected two protein bands at 104 and 100 kDa in the two cell extracts and allowed specific immunohistochemical staining in fis h brain and pituitary. For the first time in fish, these antibodies wi ll allow analysis of GR expression in different cortisol target tissue s. (C) 1998 Academic Press.