DNA binding of glucocorticoid receptor protein A fusion proteins expressed in E. coli.
In an effort to obtain large quantities of glucocorticoid receptor (GR) protein for functional and structural studies, several truncated versions of the human glucocorticoid receptor (hGR) have been expressed in E. coli as C-terminal fusion proteins with protein A. The amount of expressed protein was between 5 and 25 mg/l in the culture. South-Western blotting was initially used to demonstrate the DNA binding capacity of fusion proteins containing the DNA binding domain of GR. The hybrid proteins were highly enriched in the insoluble fraction after cell lysis. For further purification and characterization the fusion proteins were solubilized in 8 M urea. The concentration of denaturing agent was reduced by dilution and the fusion proteins were allowed to refold. The renatured GR protein A fusion proteins bound to DNA in a nitrocellulose filter binding assay. We also show that it is possible to purify the renatured fusion protein to apparent homogeneity using a single chromatographic step on DNA-cellulose.
Bonifer
C
C
Center for Biotechnology, Karolinska Institute, Huddinge University Hospital, Sweden.
Dahlman
K
K
Strömstedt
P E
PE
Flock
J I
JI
Gustafsson
J A
JA
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D004247:DNA