The cloning and expression of the ligand-binding domains of glucocorticoid and estrogen receptors

Ginseng has been commonly used as nutraceutical for thousands of years. The major active components of ginseng are ginsenosides, a diverse group of steroidal saponins, which demonstrate the ability to target a myriad of tissues, producing an array of pharmacological responses. Recent studies show the biological effects resulting from structural similarities between ginsenosides and steroids, especially, more findings on the interaction between ginsenosides and the ligand binding domain (LBD) of steroid hormone receptors. For example, Rg1 from protopanaxatriol (PPT) family are functional ligand of glucocorticoid receptor (GR); while Rb1 from protopanaxadiol (PPD) family acts as phytoestrogen to estrogen receptorbeta (ERbeta). To give the novel insight into the basic mechanisms of steroid hormone receptors-ginsenosides interaction that control "the ginsenoside induced steroid hormone receptor" signaling pathways, it is necessary to study the crystal structures of ginsenoside and LBD. However, the production of soluble functional receptors in E. coil has generally accepted to be difficult. Thus, large-scale expression and purification of LBD with binding ability represents a rate-limiting step in this area of research. In this study, we report the expression, purification, and characterization of the functional LBD of hGR/hERalpha/hERbeta.;hGR-LBD was expressed using GST tag with a single point mutation of F602S to overcome its insolubility problem. Although hGR-LBD was expressed in E.coli at high level, an appropriate condition to express it as soluble form still couldn't be found after a wide range of conditions have been attempted. To resolve the problem, hGR-LBD inclusion bodies were refolded by N-lauroylsarcosine (SKL) solubilization followed by dialysis. Finally, hGR-LBD inclusion bodies were successfully renaturated with ligand binding capability.;LBDs of hERalpha and hERbeta were expressed in E.coli both as soluble form and inclusion bodies. Due to the sensitivity of protein to oxidation and aggregation, the inclusion bodies of hERalpha/hERbeta-LBD were prepared for further purification. After a denaturation-renaturation process, a single purification step using estradiol-Sepharose 6B affinity beads was performed to purify hERalpha/hERbeta-LBD. The bound hERalpha/hERbeta-LBD was treated with iodoacetic acid to S-alkylate all the free cysteines before elution. The purified hERalpha/hERbeta-LBD after a simple dialysis was used for crystallization assays. Microcrystals were obtained in about 20 crystallization screening conditions, which confirmed that the prepared hERalpha/hERbeta-LBD met the high purity and homogeneity requirements for crystal growth.;In summary, my studies present new strategies to express, refold and purify LBDs of hGR/hERalpha/hERbeta with binding ability. These contributed to solve the crystal structures of ginsenoside and LBD.