Journal Article Expression and characterization of the Renilla luciferase with the cumulative mutation

Matsujiro, Ishibashi(鹿児島大学農学部)  ,  Ryo, Kawanabe(鹿児島大学農学部)  ,  Norie, Amaba(鹿児島大学農学部)  ,  Arai, Shigeki  ,  Fina, Amreta Laksmi(鹿児島大学農学部)  ,  Kenta, Komori(鹿児島大学農学部)  ,  Tokunaga, Masao

145pp.39 - 44 , 2017-12 , Elsevier Inc.
ISSN:1046-5928
Description
Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [M. Shigehisa et al., Protein Eng. Des. Sel. 30 (2017) 7–13] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability. Furthermore, the mutant simultaneously improved a tolerance for protease digestion, e.g. trypsin and proteinase K, and for organic solvent. Residual activity of the mutant after treatment with 10% 2-propanol, 10% DMF and 20% DMSO at 35 °C for 1 h was 29.4, 24.8 and 91.3%, respectively, whereas that of the wild type was 0.4, 0.1 and 24.3%, respectively.

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