Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E. coli.
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.
Eul
J
J
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Faculté de Médecine, Strasbourg, France.
Meyer
M E
ME
Tora
L
L
Bocquel
M T
MT
Quirin-Stricker
C
C
Chambon
P
P
Gronemeyer
H
H
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
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D011397:Promegestone