Protelytic sensitivity comparison of native and R330Q mutant luciferases

Introduction: Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg2+; known to be most efficient bioluminescence system that makes it an excellent tool for reporter in nano-system biology and medicine. In spite of wide ranges of luciferase application, it is unstable against proteolytic degradation that reduces its half-life, and therefore leads to loss in sensitivity and precision in analytic applications. Materials and methods: In order to generate more stable luciferase against protease digestion, we substituted a tryptic site, arginine 330 with glutamine. The aim of this study was the expression and purification of this mutant and comparison of its stability against trypsin digestion with wild type luciferase. The Quik-change site-directed mutagenesis method is performed and after that mutant enzyme, R330Q, under different conditions such as temperature, time and concentration of lactose was successfully expressed and then by Ni-NTA sepharose column chromatography purified. Proteolysis conditions of this mutant and native enzymes investigated. Results: The results of proteolysis experiments showed that the sensitivity of this mutated enzyme R330Q has not considerable difference compared to the wild type, indicating the mutated enzyme is sensitive to the serine protease trypsin. Conclusion: Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation. In order to Improvement of luciferase stability against trypsin, mutagenesis was performed by SDM. The results showed that the mutant enzyme is not resistant to the trypsin protease

کلیدواژه ها:

Luciferase ، Mutation ، R330Q ، Proteolysis ، Trypsin