INVESTIGADORES
ERIJMAN Leonardo
artículos
Título:
Purified Estrogen Receptor DNA Binding Domain Expressed in Escherichia coli Activates Transcription of an Estrogen-responsive Promoter in Cultured Cells
Autor/es:
NARDULLI, A.M.; LEW, D.; ERIJMAN, L.; SHAPIRO, D.J.
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Lugar: Rockville, MD; Año: 1993 vol. 266 p. 24070 - 24076
ISSN:
0021-9258
Resumen:
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The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA bind ing domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expres sion system. Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequencespecific binding to a DNA fragment containing a consensus estrogen response ele ment. The DBD protein has been purified to near ho mogeneity. Determination of the rotational relaxation time of the dansylated DBD by fluorescence polariza tion and size fractionation by Superdex column chro matography indicate that the DBD is a monomer in solution. The DBD forms a single proteinestrogen re sponse element complex in gel mobility shift assays at DBD concentrations of 183,600 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 devel oped a simple liposomebased system for delivery of protein into cultured cells. Transfected DBD protein elicited large, concentrationdependent increases in transcription of an estrogen receptor regulated re porter gene. These data demonstrate that the bacte rially expressed DNA binding domain, which repre sents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate tran scription of an estrogenresponsive promoter in ver tebrate cells.
