Novel and simple two-step purification of a full-length rat glucocorticoid-receptor expressed in a baculovirus system.
We purified the activated recombinant glucicorticoid receptor (GR) overexpressed in insect cells by sequential chromatographies using Mono Q and Mono S columns. This procedure was based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH (pH 8.4). The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. The purified GR was able to bind specifically to a DNA fragment containing the glucocorticoid response element. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.
Hyodo
M
M
Department of Biochemistry, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan.
Okamoto
K
K
Shibata
K
K
Suematsu
N
N
Isohashi
F
F
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins