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Proceedings Paper

Engineering luciferase enzymes and substrates for novel assay capabilities
Author(s): Keith V. Wood
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Paper Abstract

In the development of HTS as a central paradigm of drug discovery, fluorescent reporter molecules have generally been adopted as the favored signal transducer. Nevertheless, luminescence has maintained a prominent position among certain methodologies, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a broader range of applications due to their sensitivity, extensive linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by development several new assay designs for diverse targets such as kinases, cytochrome p450's, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for drug discovery, highlighting new detection capabilities brought about by engineering luciferase enzymes and substrates. In reporter gene applications, modified luciferases have provided greatly improved expression efficiency in mammalian cells, improved responsiveness to changes of transcriptional rate, and increased the magnitude of the reporter response. Highly stabilized luciferase mutants have enabled new assays strategies for high-throughput screening based on detection of ATP and luciferin. Assays based on ATP support rapid analysis of cell metabolism and enzymatic processes coupled to ATP hydrolysis. Although luciferin is found natively only in luminous beetles, coupled assays have been designed using modified forms of luciferin requiring the action of second enzyme to yield luminescence. Due to the very low inherent background and protection of the photon-emitter afforded by the enzyme, bioluminescent assays often outperform the analogous fluorescent assays for analyses performed in multiwell plates.

Paper Details

Date Published: 22 June 2004
PDF: 9 pages
Proc. SPIE 5328, Microarrays and Combinatorial Techniques: Design, Fabrication, and Analysis II, (22 June 2004); doi: 10.1117/12.528447
Show Author Affiliations
Keith V. Wood, Promega Corp. (United States)


Published in SPIE Proceedings Vol. 5328:
Microarrays and Combinatorial Techniques: Design, Fabrication, and Analysis II
Dan V. Nicolau; Ramesh Raghavachari, Editor(s)

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