Abstract

This chapter explores the history of the bioengineering advances that have been applied to common luciferase enzymes and the improvements that have been accomplished by this work. The primary focus is placed on firefly luciferase (FLuc), Gaussia luciferase (GLuc), Renilla luciferase (RLuc), Oplophorus luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux). Beginning with the cloning and exogenous expression of each enzyme, their step-wise modifications are presented and the new capabilities endowed by each incremental advancement are highlighted. Using the historical basis of this information, the chapter concludes with a prospective on the overall impact these advances have had on scientific research and provides an outlook on what capabilities future advances could unlock.

Highlights

  • 1.1 Historical perspective on the discovery of luciferase enzymesThe bioluminescent phenotype, which is spread across a variety of different insects, bacteria, fungi, and marine animals, has intrigued mankind since before the dawn of the modern scientific era [1]

  • Following the exogenous expression of the previously described firefly and bacterial luciferases, Renilla luciferase was isolated from the sea pansy Renilla reniformis [7] and Oplophorus luciferase was isolated from the deep-sea shrimp, Oplophorus gracilirostris [8]

  • This study showed that the split Renilla luciferase (RLuc) reporter signal could be modulated by using an inducible promoter (e.g., NFκB promoter/enhancer) to regulate the expression level of one of the two fragments

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Summary

Historical perspective on the discovery of luciferase enzymes

The bioluminescent phenotype, which is spread across a variety of different insects, bacteria, fungi, and marine animals, has intrigued mankind since before the dawn of the modern scientific era [1]. The discovery that proteins, which would come to be known as luciferases, were responsible for bioluminescent production can be traced to early experiments by Raphael Dubois, who was able to produce bioluminescence in situ by mixing the contents of click beetle abdomens in cold water and extracting the components required for light production [2]. It was not until the late 1940s that the first luciferase protein was successfully purified from fireflies [3]. This was recently changed with the introduction of fungal luciferase as a novel luciferase system, which like bacterial luciferase is capable of genetically encoding both the luciferase and luciferin pathway genes to support autobioluminescent production [12]

Available luciferase systems for biotechnological applications
The necessity of engineering luciferase proteins
Common methods for engineering improvements
Background
Initial application and limitations
Engineering improved expression and output
Engineering alternative output wavelengths
Engineering alternative signal kinetics
Engineering split luciferase applications
Engineering split and paired luciferase applications
Initial uses and limitations
Engineering eukaryotic expression
Engineering increased light output
Engineering improved bioreporter functionality
Potential future engineering goals
Outlook for future developments
Findings
Conclusion
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