Stabilization of luciferase from Renilla reniformis using random mutations

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Renilla luciferase (RLuc) is a valuable bioluminescent reporter enzyme.
• The wild-type RLuc exhibits limited stability in acidic conditions and at moderate temperatures.
• Enhancing RLuc stability is crucial for broader applications in molecular biology and diagnostics.

Purpose of the Study:

• To improve the stability of Renilla luciferase (RLuc) through protein engineering.
• To identify specific mutations that confer enhanced thermal and chemical stability to RLuc.
• To characterize the biochemical properties of engineered RLuc mutants.

Main Methods:

• Expression and purification of wild-type RLuc in Escherichia coli.
• Random mutagenesis of the Rluc gene using error-prone PCR.
• Screening of mutants for luminescence at an elevated temperature (34°C).
• Characterization of mutant RLuc proteins including solubility, specific activity, and thermal stability assays (thermal shift assays).

Main Results:

• Three stable RLuc mutants (N264SS287P, N178D, F116LI137V) were successfully generated.
• Mutants exhibited higher solubility and specific activity compared to the wild-type RLuc.
• The N264SS287P mutant showed enhanced stability at approximately 5°C higher than the wild type.
• The F116LI137V mutant displayed altered denaturation kinetics, starting lower but ending higher than wild-type.

Conclusions:

• Random mutagenesis is an effective strategy for engineering enhanced stability in Renilla luciferase.
• The identified mutations provide insights into RLuc structure-function relationships and stability mechanisms.
• These engineered RLuc variants offer improved performance for various biochemical and biotechnological applications.