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.