Design of disulfide bridge as an alternative mechanism for color shift in firefly luciferase and development of secreted luciferase

Abstract

The bioluminescence reaction, which uses luciferin, Mg²⁺-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralisfirefly luciferase to make three separate mutant enzymes with a single bridge. Moreover, C⁸¹-A¹⁰⁵C mutant luciferase was modified and successfully secreted to the extracellular medium. By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red and the optimum temperature of activity was also increased (up to 10 °C more than wild type). Amongst mutants with a disulfide bridge, P⁴⁵¹C-V⁴⁶⁹C and L³⁰⁶C-L³⁰⁹C mutants exhibit a single peak in the red region of the spectrum at pH 7.8. It is worthwhile to note that with the design of a secreted luciferase, the increased optimum temperature, thermostability and emission of red light might make mutant luciferase suitable reporters for the study of gene expression in high through-put screening.