A New Lineage of Artificial Luciferases for Mammalian Cell Imaging

The present protocol introduces a new lineage of artificial luciferases (ALucs) with unique optical properties for mammalian cell imaging. The primary candidate sequence was first created with a sequence logo generator, resulting in a total of 11 sibling sequences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools in public databases. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set. The protocol also exemplifies that the new lineage of ALucs was strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.