Methods and composition for measuring the amount of vitamin D derivatives
Methods and compositions for measuring the amount of vitamin D derivatives are disclosed. Fluorescence Resonance Energy Transfer (FRET) in combination with a modified ligand-binding domain of the vitamin D receptor (LBD-VDR) to measure vitamin D derivatives are also disclosed.
1. A polypeptide comprising a modified VDR ligand-binding domain (VDR-LBD) and a donor-acceptor pair of fluorophores, wherein said polypeptide comprises at least one mutation causing said modified VDR to have a higher binding affinity to 25-hydroxyvitamin D relative to calcitriol, and wherein said at least one mutation comprises a substitution at amino acid position 274, 147, 150, 227, 230, 233, 234, 271, 275, 286, 288, 300, 309, 313, 418, 237, 143, 278, 305 and 397 of SEQ ID NO: 1.
2. The polypeptide of claim 1 wherein said mutation at position 274 comprises a substitution of a basic amino acid residue for an aliphatic amino acid residue.
3. The polypeptide of claim 1 wherein said at least one mutation comprises Arg274Leu.