Purification of Vitamin D Receptor for Crystallization

Vitamin D, through its intracellular receptor, plays a major role in mineral homeostasis, boneremodeling, cell growth, and differentiation. It also displays immune and anticancer actions. Thisproject will determine the structure of the vitamin D-3 receptor (VDR), both with and without its naturalhormone, 1,25-dihydroxy vitamin D-3 (1,25(OH)2D3). To achieve this goal, recombinant VDR will beexpressed and purified by a scheme that will be developed to optimize yield and purity. The expressionsystem of choice is a yeast host with a copper-inducible ubiquitin-VDR vector that has been optimizedto produce 0.3 -- 3.0 percent of total soluble protein as VDR. This system has been shown to yieldintact, nonproteolyzed VDR that retains its native affinity for both 1,25(OH)2D3 and DNA. The plan isto devise a purification scheme utilizing the incorporation of a histidine tag in the VDR to facilitate itspurification and yield. Preliminary experiments show that this his-VDR protein functions identically tonative VDR. Also, the hormone-binding domain of the VDR (VDR-HBD) will be delineated to determinethe lowest molecular weight protein that retains native affinity for the hormone. This molecule also willbe tagged with histidine and produced in the yeast system, its expression levels and function assessed,and then purified to homogeneity in milligram amounts by metal affinity chromatography for both crystalstructure and NMR solution structure determinations. The project also will include a random screen forVDR agonists and/or antagonists, and compounds that are active without displaying hypercalcemicproperties would be candidates for phase II structure determinations with the purified receptors to aidin rational drug design. The 1,25(OH)2D3-like drugs have potential in the treatment of diseases suchas osteoporosis, psoriasis, breast and prostatic cancer, and leukemia.