Purification and stabilization of transcriptionally active glucocorticoid receptor.
A major obstacle to the purification of glucocorticoid receptor (GR) is the very high nonspecific surface adsorption of this protein.  This phenomenon is a property of the GR itself and does not reflect overall protein concentration or buffer conditions.  We have observed that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) is unique in its ability to stabilize the receptor and largely eliminate loss to nonspecific adsorption.  We have coupled this observation with a two-step purification method that allows efficient purification and stabilization of transcriptionally active glucocorticoid receptor. For this procedure, the GR first undergoes a major purification by anion exchange chromatography following hormone binding and on-column receptor transformation. Second, the GR is resolved to homogeneity utilizing a hydrophobic interaction chromatography step which consists of a 2.5 M to 0 M NaCl gradient elution of contaminating proteins followed by displacement of GR by CHAPS.  GR at both stages of purification was able to activate transcription from the glucocorticoid response element containing the promoter region of the long terminal repeat of the mouse mammary tumor virus.  This simple and efficient methodology should be of a considerable advantage for studies of the biology of the active, full-length GR.
Warren
B S
BS
Hormone Action and Oncogenesis Section, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
Kusk
P
P
Wolford
R G
RG
Hager
G L
GL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic; D014222:Triamcinolone Acetonide / Q000378:metabolism
D015345:Oligonucleotide Probes; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D014222:Triamcinolone Acetonide