Development of a highly sensitive in vitro system to detect and discriminate between vitamin D receptor agonists and antagonists based on split-luciferase technique

Publication date: Available online 1 November 2017
Source:The Journal of Steroid Biochemistry and Molecular Biology
Author(s): Hiroki Mano, Shinichi Ikushiro, Nozomi Saito, Atsushi Kittaka, Toshiyuki Sakaki
Split-luciferase techniques are widely used to detect protein-protein interaction and bioactive small molecules including some hormones and vitamins. Previously, we successfully expressed chimeric proteins of luciferase and the ligand binding domain (LBD) of the vitamin D receptor (VDR), LucC-LBD-LucN in COS-7 cells. The LucC-LBD-LucN biosensor was named split-luciferase vitamin D biosensor (SLDB). This biosensor can detect and discriminate between VDR agonists and antagonists in mammalian cells. In this study, we established an in vitro screening system for VDR ligands using the SLDB proteins expressed in Escherichia coli (E. coli) cells. Our in vitro screening system using cell lysate of recombinant E. coli cells could be completed within 30min, and its activity was unchanged after 10 freeze-thaw cycles. This highly sensitive and convenient system would be quite useful to screen VDR ligands with therapeutic potential for various bone-related diseases, age-related cognitive disorders, cancer, and immune disorders. In addition, our system might be applicable to diagnostic measurement of serum concentrations of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3.



http://ift.tt/2iz59hU