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Genetic and biomolecular engineering are two of the leading disciplines in biotechnology that have lead to great advancements in protein engineering. Applications of analytical bioluminescence, such as genetic reporter assays, optical in vivo imaging, and cell viability assays can often be improved by enhancing wild-type bioluminescent systems. The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. Specifically, a wide range of bioluminescent proteins and photoproteins have been engineered that can be utilized in many detection and diagnostic applications. Herein, we focus on the improvements of two of the most commonly studied photoproteins, aequorin and obelin, and their uses in a variety of bioanalytical applications. Techniques such as random mutagenesis, site-directed mutagenesis, bioluminescence resonance energy transfer, and the incorporation of coelenterazine analogues are discussed as ways that have expanded the palette of these designer proteins by altering their emission wavelengths and/or half-lifes. Strategic amino acid substitutions and insertions have been also used to improve luciferase stability in high temperature, extreme pH, and harsh chemical environments, and to customize their kinetic properties and bioluminescence colors. As researchers advance engineering techniques to expand the array of photoproteins, luciferases from fireflies, click beetles, marine organisms and bacteria, their use in bioanalytical applications will continue to grow and it is envisioned that photoproteins and bioluminescent proteins will become as diverse as their fluorescence counterparts.

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