Bright mutant beetle luciferases: performance in bioluminescence detection in live mammalian cells and insights into their spectral mechanism by tagging them with fluorescent proteins

ABSTRACT

Beetle luciferases catalyze a bioluminescent reaction with D-luciferin and ATP as substrates. Photinus pyralis firefly luciferase (luc2) is widely used as a reporter gene to monitor gene expression and for live cell imaging. However, some newer beetle luciferases (e.g., Eluc) outperform it in brightness. A number of luciferases became available that utilize cœlenterazine (CTZ) analogs as substrates and achieve higher brightness in many applications. However, there is little data that directly compares the performance of these luciferases and their specific enzyme activity in live cells. We report brighter, improved luc2 mutant variants with superior performance to luc2 and Eluc in live cell applications. By using mVenus and mNeonGreen fluorescent protein tags to directly assess reporter protein levels in live cells, we have quantitatively compared the performance of several promising beetle luciferases between each other and relative to the brightest CTZdependent luciferase, Nanoluc. Furthermore, we have discovered an atypical bioluminescence resonance energy transfer from beetle luciferases to mVenus and mNeonGreen: it shifts the light emission spectrum to shorter instead of longer wavelengths. This gives new important insights into the mechanism and unsolved questions of the light emission of beetle luciferases.