Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging

This study demonstrated a novel construction of bifunctional enzyme system to improve the luminescence generation of firefly luciferase, by bringing in a luciferin-regenerating enzyme (LRE) fusion expressed to the C terminal of luciferase, between which were connected with peptide linker. The fusion protein constructed with typical type of linker, rigid linker (EAAAK) and flexible linker (GGGGS), were analyzed comparing with the unlinked free enzyme. In vivo and in vitro assessment of the bioluminescence intensity and decaying rate to the series of Fluc-LRE enzyme complex were assayed. The fInding demonstrated that the presence of LRE remarkably enhance the generation of luminescence and remained significant stronger signal than that of the control, and the peptide-linked dual enzyme present more stability and continuation on the signal generation and lower decaying rate on signal recession, especially at low dose of Fluc injection. With the advantage of luminescence intensity and reaction period, the peptide mediated fusion expressed LRE may expand the application of Firefly luciferase on bioluminescence imaging.