title: Stabilization of luciferase from Renilla reniformis using random mutations.
abstract: 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.
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Shigehisa
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Norie
authors.initials: N
authors.lastname: Amaba
authors.affiliation: Quantum Beam Science Research Directorate, National Institutes for Quantum and Radiological Science and Technology, 2-4 Shirakata, Tokai, Ibaraki 319-1106, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Arai
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Higashi
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kawanabe
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Matsunaga
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Fina Amreta
authors.initials: FA
authors.lastname: Laksmi
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan matu@chem.agri.kagoshima-u.ac.jp.
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
mesh_terms: D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D017433:Protein Structure, Secondary; D012995:Solubility; D013696:Temperature
chemical_list: D049410:Luciferases, Renilla
keywords: Renilla reniformis; error-prone PCR; luciferase; mutation; stabilization