| Autor: |
Gedi V; Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea., Kim EH; Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea., Oh B; Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea., Kim YP; Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea.; Research Institute for Convergence of Basic Science, Hanyang University, Seoul 04763, Republic of Korea.; Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea.; Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul 04763, Republic of Korea. |
| Abstrakt: |
Gaussia luciferase ( G Luc) 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 G Luc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in G Luc (designated as G Luc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the G Luc5-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 G Luc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications. |
