Advanced Bioluminescence Reporter with Engineered

Gaussia luciferase (GLuc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type GLuc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in GLuc (designated as GLuc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the GLuc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in GLuc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications.