Firefly Luciferase Mutant with Enhanced Activity and Thermostability

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Firefly luciferase (Photinus pyralis, Ppy) is a reporter gene, but its stability and intracellular substrate (d-luciferin) levels limit applications.
• Protein engineering has yielded Ppy luciferase mutants with enhanced thermostability, pH tolerance, and catalytic efficiency.

Purpose of the Study:

• To develop a Ppy luciferase mutant with improved thermostability and enhanced luminescence at low d-luciferin concentrations for reporter gene applications.

Main Methods:

• Combined beneficial amino acid mutations from previously identified thermostable (Mutant E) and catalytically active (LGR) Ppy luciferase variants.
• Created a novel mutant (YY5) with eight amino acid substitutions compared to wild-type Ppy luciferase.

Main Results:

• The YY5 mutant demonstrated significantly improved thermostability compared to wild-type Ppy luciferase.
• YY5 exhibited brighter luminescence, particularly under conditions of limited intracellular d-luciferin availability.
• The engineered luciferase maintained yellow-green light emission (peak at 560 nm).

Conclusions:

• The YY5 mutant represents a significant advancement in Ppy luciferase engineering.
• Its enhanced stability and luminescence efficiency make it a valuable tool for reporter gene assays.
• YY5 holds potential for overcoming limitations in current biological applications of firefly luciferase.