Janaki Narahari

Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays

Luciferase-based reporter assays are powerful tools for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. Here we describe the characterization and use of a luciferase reporter enzyme derived from the marine copepod Metridia luciferase family, referred to as TurboLuc luciferase (TurboLuc). To develop TurboLuc, the wild-type luciferase was modified to decrease its size, increase brightness, slow luminescent signal decay, and provide for efficient intracellular expression. To determine the enzyme susceptibility to compound inhibition and judge the suitability of using of TurboLuc as a reporter in screening assays, purified TurboLuc enzyme was screened for inhibitors using two different compound libraries. No inhibitors of this enzyme were identified in a library representative of typical diverse low molecular weight (LMW) compounds using a purified TurboLuc enzyme assay supporting that such libraries will show very low interference with this enzyme. We were able to identify a few inhibitors from a purified natural product library which can serve as useful tools to validate assays using TurboLuc. In addition to the inhibitor profile for TurboLuc we describe the use of this reporter in cells employing miniaturized assay volumes within 1536-well plates. TurboLuc luciferase is the smallest luciferase reporter enzyme described to date (16 kDa), shows bright luminescence and low interference by LMW compounds, and therefore should provide an ideal reporter in assays applied to high-throughput screening.

Abstract 3207: Monitoring changes in NF-kB pathway regulation using highly sensitive multiplex bioluminescent reporter assays

Monitoring gene activation and repression in cellular signaling pathways requires specific tools. Reporter gene assays use a technique of attaching a genetic regulatory element, or promoter, to a gene encoding for a protein with enzymatic, chromophoric or fluorescent properties. Cloned into a plasmid vector, these sequences can be transfected or transduced into animal, plant or tissue culture cells. Reporter gene expression can be measured via bioluminescent, chemiluminescent or fluorescent output to quantify the element or protein activity in the pathway affecting the regulatory element. Bioluminescent output is formed through the catalysis of a substrate and the natural enzyme luciferase. Present in many terrestrial and marine metazoans, bioluminescence is a powerful instrument for mutational analysis of gene promoters, transcription factor dynamics, and protein-protein interactions. Our study confirmed the beneficial use of sensitive bioluminescent enzymes in reporter gene assays with wide dynamic ranges and flexibility in signal identification. Mutation of several of the luciferase genes resulted in spectral shifting and narrowing of the photon reaction products relative to the wavelength of the natural state. The mutations provide the ability to spectrally resolve the modified luciferases in a single reaction. In addition, multiplex assays relying on sequential addition of substrates can be achieved through manipulation of the enzyme-substrate reaction conditions. Marine copepod and ostracod luciferase enzymes are naturally secreted, and reporter assays using these genes can capture real-time or time course data by sampling the media of transfected cells or blood from transgenic animals and assaying for luciferase. The distinct advantage of these assays is that they are not destructive to the cells or animals. Our study used spectral separation using Gaussia/firefly and multiplex assays relying on sequential addition of substrates and Gaussia/Cypridina to monitor changes in NFkB activity in response to small-molecule agonists. Our results demonstrate the usefulness of dual-secreted luciferase assays for sensitive real-time monitoring of NFkB activity in media. Furthermore, these assays enable simultaneous detection of spectrally resolvable luciferases using filter-based detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3207. doi:1538-7445.AM2012-3207