Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
J Steroid Biochem Mol Biol. 2018 Apr;178:55-59. doi: 10.1016/j.jsbmb.2017.10.024. Epub 2017 Nov 1.
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 D and 1α,25-dihydroxyvitamin D.
荧光素酶酶切技术广泛用于检测蛋白质-蛋白质相互作用和生物活性小分子，包括一些激素和维生素。之前，我们成功地在 COS-7 细胞中表达了荧光素酶和维生素 D 受体（VDR）配体结合域（LBD）的嵌合蛋白 LucC-LBD-LucN。LucC-LBD-LucN 生物传感器被命名为荧光素酶维生素 D 生物传感器（SLDB）。该生物传感器可在哺乳动物细胞中检测和区分 VDR 激动剂和拮抗剂。在这项研究中，我们使用在大肠杆菌（E. coli）细胞中表达的 SLDB 蛋白建立了一种用于 VDR 配体的体外筛选系统。我们使用重组大肠杆菌细胞裂解物的体外筛选系统可以在 30min 内完成，并且在 10 次冻融循环后其活性没有变化。该高度敏感和方便的系统将非常有助于筛选具有治疗潜力的各种与骨骼相关疾病、与年龄相关的认知障碍、癌症和免疫紊乱相关的 VDR 配体。此外，我们的系统可能适用于血清中 25-羟维生素 D 和 1α，25-二羟维生素 D 浓度的诊断测量。
