Abstract
The inability to obtain sufficient quantities of purified glucocorticoid receptor (GR) causes difficulties in studying in vitro the interactions of GR with other proteins which contribute to the transcriptional activation of chromatin. Thus, we overexpressed the DNA-binding domains (DBD) of GR in E. coli using glutathione S-transferase (GST)-fusion protein expression vector. The DBD-GST protein (about 57 kDa), recovered in the soluble fraction, was purified by glutathione-agarose column. However, during the chromatography, more than 90% of the DBD-GST protein was cleaved into three species with molecular sizes of about 40, 35 and 30 kDa. The 40 kDa and 35 kDa proteins were immunostained with the anti-GR antibody, and the 30 kDa protein was stained with anti-GST antibody. Among these proteins, only the 57 kDa DBD-GST protein bound to the GRE-agarose. These data indicate that the properly folded 57 kDa protein has a DNA binding function and is resistant to intrinsic cleavage of the protein.
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