Expression and functional analysis of steroid receptor fragments secreted from Staphylococcus aureus.
Fragments of the DNA-binding domain of the rat glucocorticoid receptor (rGR) and the human estrogen receptor (hER) were expressed in Staphylococcus aureus as a chimeric fusion to protein A by using a modified expression vector with an artificial factor X-cleavage site. The secreted product was isolated by hydrophobic chromatography on Phenyl-Sepharose and purified on DNA-cellulose or by anion-exchange chromatography. After cleavage of the protein A moiety, the purified rGR DNA-binding domain from amino acids 406 to 523 (rGR406-523), binds specifically to a glucocorticoid responsive element as a homodimer but cannot form heterodimers with the DNA-binding domain of the hER. Amino acids 510 to 523 following the zinc finger region, as well as free sulfhydryl-groups are necessary for DNA-binding, which is more efficient when the tripeptide Gly-Gly-Cys is added to the carboxy terminal end. Despite its specific interaction with DNA, rGR406-523 does not activate transcription from the MMTV promoter in a cell-free system that efficiently responds to addition of native GR, suggesting that regions essential for transcriptional activation in vitro are located outside of the DNA-binding domain.
Cao
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X
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Marburg, Germany.
Preiss
T
T
Slater
E P
EP
Westphal
H M
HM
Beato
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M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics*; D015533:Transcriptional Activation
D010446:Peptide Fragments; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D004247:DNA