Ho, Pei-I, Casalena, Dominick and Auld, Douglas (2018) Characterization and use of Turbo Luciferase as a reporter for high-throughput assays. Biochemistry. ISSN 10.1021/acs.biochem.8b00290

Abstract

Abstract: 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 reporter enzyme derived from the marine copepod Metridinidae luciferase family, referred to as TurboLuc™. To develop TurboLuc luciferase, 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 (16kDa), shows bright luminescence, and low interference by LMW compounds and therefore should provide an ideal reporter in assays applied to high-throughput screening.

Item Type:	Article
Date Deposited:	24 Jul 2018 00:45
Last Modified:	24 Jul 2018 00:45
URI:	https://oak.novartis.com/id/eprint/35553