Abstracts

The development of luciferase applications requires small size, high expression level, and bright emission. In recent years, small bright luciferases such as ALuc (Artificial luciferase) (21 kDa), NanoLuc (19 kDa), GLuc (Gaussia luciferase) (18 kDa) and TurboLuc (16 kDa) have been widely used because their small size can reduce the misfolding and the steric hindrance, and they have a superiority for bioluminescence energy transfer (BRET) based assay due to the short distance between luciferase and fluorescent protein. Recently, we serendipitously discovered an ALuc deletion mutant that retained high luminescence activity. We then focused on minimizing ALuc, and finally succeeded in producing the smallest luciferase (120 aa, 13 kDa), named “picALuc” (a coined word of “pica” and “ALuc”, where the beginning pica includes the meanings of pico (small) and pika (the onomatopoeia of light and flash in Japanese)). Surprisingly, picALuc completely retained the luminescence activity of full-length ALuc, and the highest luminescence intensity was almost equal to that of NanoLuc when secreted from mammalian cells. In this work, we engineered novel picALuc variants. First, the original picALuc luminescence was still dim compared to other bright luciferases in terms of expression in Escherichia coli. We improved the activity through charged oligopeptide fusion, resulting in up to approximately 50-fold increase in luminescence intensity. We named the new version of picALuc “picALuc2.0”. Second, many luciferases exhibit flash-type luminescence patterns, in which the lights rapidly fade immediately after the substrate addition. We found that single-residue deletion mutants of picALuc exhibited longer luminescence lifetimes (glow-type luminescence), could be reactivated multiple times upon re-addition of substrate, and could be reused for day-scale time at room temperature. We named this version of picALuc “picARe” (representing “shining” in Japanese), after the words such as reuse, recycle and revival. Currently, we have been improving higher luminescence activity, long-lasting luminescence, and color change through protein engineering, adjustment of the reaction solution composition, and use of substrate analogues. These further improvements will open new doors in the luciferase world.