随机突变稳定Renilla reniformis荧光素酶

M. Shigehisa, Norie Amaba, S. Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, F. A. Laksmi, M. Tokunaga, M. Ishibashi
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引用次数: 12

摘要

我们在大肠杆菌中表达了Renilla reniformis的荧光素酶(RLuc)。用Ni-NTA柱纯化RLuc并对其进行表征。它在酸性溶液和30°C时不稳定。为了增加RLuc的稳定性,采用易出错的聚合酶链反应随机突变RLuc基因。通过在含有底物coelenterazine的平板上检测34℃下的发光来筛选携带突变基因的大肠杆菌。获得了N264SS287P、N178D和F116LI137V三个突变体。这些突变体的溶解度和比活性均高于野生型。此外,N264SS287P突变体在比野生型高约5℃的温度下保持稳定,而F116LI137V突变体的变性开始温度比野生型低5℃,结束温度比野生型高7℃。在本研究中,我们使用热移法和计算机程序来检查获得的突变。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Stabilization of luciferase from Renilla reniformis using random mutations
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.
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