Development and Application of a Brighter Chemiluminescent Protein for Bioimaging

Abstract

Fluorescent protein revolutionized our understanding of biological processes. However the requirement for external illumination definitely precludes its universal application to certain biological processes. On the other hand, bioluminescent proteins such as luciferase don’t require the external illumination, thereby bioluminescence can be imaged without phototoxicity and auto-fluorescence from the specimen, allowing us to detect signals with high signal-to-noise ratio. These properties make bioluminescent proteins potentially superior to fluorescent proteins as a bioimaging tool. However, existing bioluminescent proteins are too dim to be measured in real time, requiring longer exposure than fluorescence imaging that takes less than 1 second. To overcome this drawback, we conducted random mutagenesis on Renilla reniformis luciferase (Rluc) to improve the intensity. Then, the luminescence intensity was further increased by fusion of the improved Rluc to a yellow fluorescent protein with high FRET efficiency. The chimeric protein named ‘Nano-lantern’ showed much brighter luminescence than the commercially available Rluc, enabling not only real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence but also sensitive tumor detection in freely moving unshaved mouse which has never been possible before. This super-duper luminescent protein will revolutionize conventional bioimaging by allowing visualization of biological phenomena not seen before.