title: Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E. coli.
abstract: Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively. The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.5 nM and was specifically photocross-linked with the synthetic progestin R5020 in crude bacterial lysates. Labelling of intact bacterial cells with [3H]R5020 revealed that the majority, if not all, of the bacterially produced hormone-binding domain was active. No differences in the binding to a synthetic palindromic glucocorticoid/progestin-responsive element (GRE/PRE) were found when the bacterially produced cPR DNA-binding domain was compared in methylation interference assays with the full-length chicken progesterone receptor form A expressed in eukaryotic cells. The study of dissociation kinetics, however, revealed differences in the half-life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA-binding domain. DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific 'footprints' on the mouse mammary tumour virus long terminal repeat (MMTV-LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor.
authors.affiliation: Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Faculté de Médecine, Strasbourg, France.
authors.forename: J
authors.initials: J
authors.lastname: Eul
authors.forename: M E
authors.initials: ME
authors.lastname: Meyer
authors.forename: L
authors.initials: L
authors.lastname: Tora
authors.forename: M T
authors.initials: MT
authors.lastname: Bocquel
authors.forename: C
authors.initials: C
authors.lastname: Quirin-Stricker
authors.forename: P
authors.initials: P
authors.lastname: Chambon
authors.forename: H
authors.initials: H
authors.lastname: Gronemeyer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D011374:Progesterone / Q000378:metabolism; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D011397:Promegestone