Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system.
We have expressed a full-length human glucocorticoid receptor (hGR) in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system (BEVS). The level of expression is approximately 100-fold greater than in CEM-C7 cells. Between 0.5-1.0 mg hGR can be generated per liter of Sf9 cell culture. The expressed hGR is capable of binding glucocorticoids with specificity and high affinity. Covalent labeling with 3H-dexamethasone mesylate and Western blot analysis using a polyclonal antibody indicate that the molecular weight of the expressed protein is approximately 94 k. The nonactivated receptor sediments as a 8-9S complex in sucrose gradients and can be heat activated to a 4S form. The activated receptor is capable of retarding the migration of a 23 base-pair DNA fragment containing the glucocorticoid response element from the tyrosine aminotransferase gene. These data indicate that the expressed GR displays characteristics identical to those of GR from mammalian cells. By scaling up this culture we can, for the first time, obtain enough purified full-length receptor for crystallographic and functional studies which could provide new insight into exactly how hGR works.
Srinivasan
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Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Thompson
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EB
D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000378:metabolism; D004267:DNA Viruses / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology; D005938:Glucocorticoids / Q000378:metabolism / Q000502:physiology; D007814:Larva / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D007915:Lepidoptera / Q000166:cytology*; D009036:Moths / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D011233:Precipitin Tests; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*
D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D004247:DNA