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Luciferase from the North American firefly Photinus Pyralis catalyzes a highly efficient bioluminescent reaction that produces yellow-green light (557 nm at pH 7.8). This bioluminescence system has been adapted for a variety of applications including gene reporter assays, detection of bacteria and toxins, whole-cell biosensor measurements, and in vivo imaging. A luciferase variant with a red-shifted bioluminescence spectra and high specific activity, two qualities that are challenging to achieve simultaneously, would be especially useful when paired with a green-emitting enzyme in a dual-color reporter assay, or used alone for improved imaging in living animals.

Our lab previously reported a red-emitting mutant named Ppy RE, which contained a single mutation at S284T that shifted the emission maximum from the 557 nm to 615 nm at pH 7.8. In a subsequent publication, Ppy RE was enhanced with five mutations to make Ppy RE-TS, which exhibited improved thermostability at 37°C, but at the expense of reducing the red-shift to 610 nm. In this report, we further improve upon Ppy RE-TS with four rational point mutations that red-shift the emission maximum to 617 nm, dramatically increase the enzyme thermostability, and improve the total light emission during 8-second assays. This new enzyme, named PRET7, is characterized and evaluated for performance in E. Coli and HEK293 mammalian cell lines. Compared to a commercially available enzyme called CBR, which has been codon-optimized for expression in mammalian cells, PRET7 produces a stronger signal over 8-second assays in which the least expensive reagents available are used. Aiming to improve the already impressive performance of PRET7, we are currently awaiting results from collaborators who are evaluating a human codon-optimized version of PRET7 in HEK293 cells.

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