title: Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies.
abstract: Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from Escherichia coli inclusion bodies. The heterologously expressed protein constructs retained full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with (2)H, (13)C, and (15)N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed.
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors: Jinge
authors: J
authors: Zhu
authors: Hongyu
authors: H
authors: Rao
authors: Marco
authors: M
authors: Tonelli
authors: William M
authors: WM
authors: Westler
authors: Kiran K
authors: KK
authors: Singarapu
authors: John L
authors: JL
authors: Markley
authors: Hector F
authors: HF
authors: DeLuca
authors: Fariba M
authors: FM
authors: Assadi-Porter
mesh_terms: D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics*; D007553:Isotope Labeling / Q000379:methods; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D008024:Ligands; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D004247:DNA; D003903:Deuterium