通过基因嵌合和随机诱变提高萤火虫荧光素酶的实用性

Kozo Hirokawa, Naoki Kajiyama, Seiji Murakami
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引用次数: 34

摘要

为了提高萤火虫荧光素酶的实用性,我们将Photinus pyralis荧光素酶与Luciola crucata荧光素酶的耐热变体进行了基因嵌合。其中一种嵌合型荧光素酶对底物ATP的Km值较低,稳定性与耐高温的十字花科植物荧光素酶相似。然后,我们在相应的基因中引入随机突变,并筛选增加的催化效率。Thr219、Val239和Val290的氨基酸置换影响了动力学参数。因此,我们将这三种突变结合起来。其中一个突变体ABcT219I (V239I)表现出与P. pyralis荧光素酶相当的高催化效率和与耐高温L. crucata荧光素酶相似的高稳定性。研究了生物发光发射光谱的ph依赖性。与迄今为止表征的野生型萤火虫荧光素酶相比,突变体没有表现出ph依赖的红光谱位移。
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Improved practical usefulness of firefly luciferase by gene chimerization and random mutagenesis

To improve the practical usefulness of the firefly luciferase, we performed gene chimerization between Photinus pyralis luciferase and a thermostable variant of Luciola cruciata luciferase. One chimeric luciferase showed low Km value for substrate ATP and similar stability to thermostable L. cruciata luciferase. We then introduced random mutations in the corresponding gene and screened for increased catalytic efficiency. Amino acid replacement of Thr219, Val239 and Val290 affected the kinetic parameters. Therefore, we combined these three mutations. One mutant, ABcT219I,V239I, showed high catalytic efficiency comparable to P. pyralis luciferase and high stability similar to thermostable L. cruciata luciferase. The pH-dependence of the bioluminescence emission spectra was also examined. In contrast to wild-type firefly luciferases characterized to date, the mutant did not show the pH-dependent red spectrum shift.

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