Development of a multiplexed bioluminescent cell-based assay with luc gene from Luciola italica for high throughput screening of cholesterol-lowering drugs

The cDNA encoding the luciferase from the Italian firefly Luciola italica has been recently cloned and preliminary expressed in E. coli The bioluminescence emission of the Italian firefly luciferase has a maximum at 566 nm at pH 7.8 and shows extended light emission decay kinetics. This new luciferase has been cloned in suitable vectors and expressed in E. coli, S. cereviesiae and mammalian cells (HepG2) to explore its applicability as reporter protein in whole-cell biosensors, non-invasive in vivo imaging and multiplexed assays. Diverse mutants were also obtained by site-directed or random mutagenesis. In the present study, a new cell-based assay with two luciferases emitting at different wavelengths has been developed to monitor in vivo the two pathways of bile acid biosynthesis. Bile acid biosynthesis is in fact a key step of intracellular cholesterol homeostasis and, in turn, cholesterol synthesis rate in hepatocytes. Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). We evaluated the ability of natural and synthetic bile acids and other compounds to activate or hamper the two bile acid synthesis pathways using a recombinant HepG2 cell-based luciferase multiplexed assay. Cells were stably transfected with the expression vector pcDNA3.1-FXR, containing the cDNA encoding human Farnesoid X Receptor, in order to obtain clones that stably expressed the receptor. The clones were transiently transfected with reporter plasmids containing the CYP7A1 promoter driving the expression of a wild-type Photinus pyralis luciferase and the CYP27A1 promoter regulating the expression of a red emitting thermostable L. italica luciferase. The developed multiplexed cell-based assay allows the simultaneous monitoring of the two pathways in a high throughput format (96-well microtiter plate), being thus suitable for the screening of new cholesterol-lowering drugs.