Bioluminescent imaging (BLI) with Firefly Luciferase is one of the best techniques for visualizing cell growth and movement <i>in vivo</i>, but there are still many ways to optimize its capabilities. To improve Firefly Luciferase for BLI, it has been subjected to various protein engineering projects. Previously, an orthogonal pair of mutant luciferases and modified substrates, named Pecan and Cashew, was developed to have minimal cross reaction. This pair is considered to be the best tool for multicomponent imaging <i>in vivo</i>, has been used in rapid imaging studies, and more. But their mutations for selectivity also caused over 20-fold decrease in brightness compared to WT. In this project, Pecan and Cashew were improved by further mutating the proteins at positions previously shown to improve brightness. Multiple mutants were discovered to be reproducibly 2- to 10-fold brighter and more selective than their parent enzymes. The resulting brightest orthogonal pair will have a better signal to noise ratio and be able to detect signal from smaller amounts of cells.

Evolving a pair of brighter luciferases for multicomponent bioluminescent imaging

ACS Spring 2022

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