Purification and Stabilization of Transcriptionally Active Glucocorticoid Receptor
| Autor: | Barbour S. Warren, Ronald G. Wolford, Philip Kusk, Gordon L. Hager |
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| Rok vydání: | 1996 |
| Předmět: |
Transcription
Genetic Molecular Sequence Data Triamcinolone Acetonide Biochemistry Cell Line Mice Cytosol Receptors Glucocorticoid Glucocorticoid receptor Chaps Mammary tumor virus Animals Promoter Regions Genetic Receptor Molecular Biology Repetitive Sequences Nucleic Acid Hormone response element Base Sequence biology Chemistry Hydrophilic interaction chromatography Mouse mammary tumor virus Promoter Cell Biology Chromatography Ion Exchange biology.organism_classification Mammary Tumor Virus Mouse Chromatography Gel Oligonucleotide Probes Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 271:11434-11440 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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