Characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system.
The structure-function relationship of the oligomeric unactivated glucocorticoid receptor is not fully understood. An essential step in the process of understanding such a relationship involves the production of large quantities of the receptor. Using a baculovirus expression system we have been able to overproduce a recombinant rat glucocorticoid receptor (rGR). A cDNA coding for the entire rGR was introduced into the genome of the wild type baculovirus, Autographa californica nuclear polyhedrosis virus, by an in vivo recombination event. Based on specific steroid binding, insect cells infected with the recombinant baculovirus expressed 1-3 x 10(6) receptor molecules/cell which is 15-45 times more than that expressed normally in a hepatocyte. The recombinant rGR expressed in insect cells is indistinguishable from the bona fide rGR with respect to immunogenic reactivity, cytoplasmic localization, sedimentation, chromatographic and electrophoretic mobility, and hormone and DNA binding. Furthermore, the recombinant rGR is expressed as a functional protein as demonstrated by its ability to specifically bind a glucocorticoid agonist, to translocate from the cytoplasm to the nucleus upon hormone-binding, and to act as a transcriptional enhancer. Pulse labeling of recombinant baculovirus-infected insect cells with 32Pi and isolation of the labeled products by immunoprecipitation demonstrated that the recombinant rGR is a phosphoprotein. Thus, the recombinant rGR expressed in insect cells is biologically active and is suitable for structural and functional analysis. A simple three-step purification procedure of the unactivated recombinant rGR is described.
Alnemri
E S
ES
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Maksymowych
A B
AB
Robertson
N M
NM
Litwack
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D007313:Insecta / Q000382:microbiology; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013256:Steroids / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D013256:Steroids; D015500:Chloramphenicol O-Acetyltransferase