Megumi Shigehisa, Norie Amaba, Shigeki Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, Fina Amreta Laksmi, Masao Tokunaga and Matsujiro Ishibashi
We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli. RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.
Keywords: Mutation, Stabilization, error-prone PCR, luciferase, Renilla reniformis
Disusun oleh: Ludya AB/Pustakawan