Bioluminescent proteins and fluorescent proteins are commonly used to investigate protein-protein interactions, protein conformational changes and various aspects of gene expression and regulation.

However, these reporters are normally used on their own, or at best in combination with only one other bioluminescent reporter molecule due spectral overlap from the molecules' luminescent signals.

New research, published as an early access article in the journal Analytical Chemistry , describes the combination of three spectrally resolved bioluminescent reporter proteins that: "paves the way for the monitoring of multiple metabolic events for high-content screening."

The research team, led by Professor Aldo Roda of Italy's University of Bologna, combined a green-emitting Photinus pyralis lucferase and a red thermostable mutant of L. italica luciferase to monitor two different bile acid biosynthesis pathways - key parts of the cholesterol homeostasis mechanism.

The luminescent signals from these reporter molecules were found to have good spectral separation and could be expressed in cells at similar levels after a transfection step.

A third reporter molecule a secreted Gaussia princeps luciferase (GLuc) was used as an internal cell vitality control.

The 'classic' bile acid formation pathway starts with a 7a-hydroxylation of cholesterol by cholesterol 7a-hydroxylase (CYP7A1) in the liver, while the 'acidic' pathway starts with the 27-hydroxylation by sterol 27-hydroxylase (CYP27A1).

Therefore, reporter plasmids harbouring the luciferases were constructed and transfected into the cells to enable the transcriptional monitoring of CYP7A1 and CYP27A1 while GLuc was incorporated as a separate internal correction that enabled cell viability to measured and the other signals to be normalised against cell viability and number.

This high content screening (HCS) assay enabled the researchers to evaluate the ability of natural and synthetic bile acids and other compounds to activate or inhibit the two bile acid synthesis pathways.

According to the authors, "the developed assay does not suffer the limitations of previous triple-reporter assays based on green-, orange- and red-emitting clickbeetle luciferases, such as errors in the deconvolution process due to overlapping emissions."