Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells.
The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA binding domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expression system. Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequence-specific binding to a DNA fragment containing a consensus estrogen response element. The DBD protein has been purified to near homogeneity. Determination of the rotational relaxation time of the dansylated DBD by fluorescence polarization and size fractionation by Superdex column chromatography indicate that the DBD is a monomer in solution. The DBD forms a single protein-estrogen response element complex in gel mobility shift assays at DBD concentrations of 18-3,600 nM, suggesting that the DBD is bound to both halves of the palindromic estrogen response element. To investigate the ability of the DBD expressed in bacteria to activate gene expression, we have developed a simple liposome-based system for delivery of protein into cultured cells. Transfected DBD protein elicited large, concentration-dependent increases in transcription of an estrogen receptor regulated reporter gene. These data demonstrate that the bacterially expressed DNA binding domain, which represents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate transcription of an estrogen-responsive promoter in vertebrate cells.
Nardulli
A M
AM
Department of Biochemistry, University of Illinois, Urbana 61801.
Lew
D
D
Erijman
L
L
Shapiro
D J
DJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D004958:Estradiol / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol; D015500:Chloramphenicol O-Acetyltransferase