Ishibashi Matsujiro, Kawanabe Ryo, Amaba Norie, Arai Shigeki, Laksmi Fina Amreta, Komori Kenta, Tokunaga Masao
Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Protein Expr Purif. 2018 May;145:39-44. doi: 10.1016/j.pep.2017.12.010. Epub 2017 Dec 28.
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 [1] 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.
来自海肾（Renilla reniformis）的荧光素酶（RLuc）作为一种报告蛋白和生物成像探针是一种很好的研究工具，它利用底物腔肠素产生蓝光。然而，由于RLuc在各种条件下都不稳定，其应用受到限制。因此，人们尝试提高RLuc的热稳定性。在本研究中，将先前报道的5个突变[1]和通过定点诱变获得的1个突变进行了组合。这种组合的结果是热稳定性效应增强，突变体的稳定性提高了约10°C。此外，该突变体同时提高了对蛋白酶消化（如胰蛋白酶和蛋白酶K）以及有机溶剂的耐受性。在35°C下用10%异丙醇、10%二甲基甲酰胺和20%二甲基亚砜处理1小时后，突变体的残余活性分别为29.4%、24.8%和91.3%，而野生型的残余活性分别为0.4%、0.1%和24.3%。
