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

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 178; pp. 55 - 59
• Main Authors: Mano, Hiroki, Ikushiro, Shinichi, Saito, Nozomi, Kittaka, Atsushi, Sakaki, Toshiyuki
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.04.2018
• Subjects: Binding Sites; Bioluminescent sensor; Biosensing Techniques; Calcifediol - pharmacology; HL-60 Cells; Humans; In Vitro Techniques; Ligands; Luciferases - genetics; Luciferases - metabolism; Protein Domains; Receptors, Calcitriol - agonists; Receptors, Calcitriol - antagonists & inhibitors; Receptors, Calcitriol - metabolism; Screening system; Split-luciferase; Vitamin D; Vitamin D receptor; Vitamins - pharmacology; Split-luciferase; Screening system; RXRα; VDR; Bioluminescent sensor; TIF2; Vitamin D receptor; LucC; LBD; LucN; CYP27B1; Vitamin D; DRIP205; SRC-1; ELISA
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2017.10.024

•Chimeric protein between split-luciferase and vitamin D receptor was constructed.•In vitro screening system for VDR ligands was established using this biosensor.•This system could be used for measurement of serum 25(OH)D3 concentration. 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.