Firefly luciferase catalyzes a redox reaction with the substrates luciferin and ATP to produce light, resulting in bioluminescence. Luciferase is used in Bioluminescent Imaging (BLI) to monitor biological processes in living systems. While the sensitivity and dynamic range of luciferase in BLI surpasses those of traditional fluorescence-based methods, BLI is constrained by the luciferase enzyme’s weak light emissions, high binding coefficient (<i>K<sub>M</sub></i>), and thermal and pH instability. Previous efforts to engineer luciferases have primarily focused on improving single biochemical properties while neglecting others. In order for BLI to reach its full potential, it is important for luciferase to have improved properties overall. Here, we report development of a plate-based screen that simultaneously measures wavelength, luciferin binding, and thermal stability. We used Statistical Coupling Analysis (SCA) of luciferase protein homologs to design mutant libraries, which were generated by synthetic shuffling. We plan to screen our libraries for multiple properties at once, and select for luciferases with optimized function for use in medical diagnostics and imaging.

Engineering luciferase to evolve improved properties

251st National Meeting (2016)

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