Thermostabilization of firefly luciferase by in vivo directed evolution
Thermostabilization of firefly luciferase by in vivo directed evolution
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DOI：
10.1093/protein/gzr044
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发表时间：
2011-11-01
影响因子：
2.4
通讯作者：
Ugarova, Natalia N.
中科院分区：
文献类型：
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作者：
Koksharov, Mikhail I.;Ugarova, Natalia N.
萤火虫荧光素酶广泛应用于生物技术和分子生物学的许多领域。然而，野生型（WT）脱氢酶在升高的温度下的快速失活常常阻碍它们的应用。一个简单的非致死性的体内筛选方案被用来确定大肠杆菌菌落中的荧光素酶的热稳定突变体。该方案允许以快速和有效的方式进行每个诱变循环。对编码Luciola mingrelica荧光素酶第130-390位氨基酸残基的部分基因进行了4轮定向进化。所得到的命名为4 TS的突变体在42 ℃下的半衰期为10小时，与WT荧光素酶相比高65倍。此外，与WT酶相比，突变体4 TS的比活性增加了1.9倍，ATP的Km降低了5.7倍，并且具有更高的最适温度。4 TS含有8个突变，其中4个突变被认为是热稳定性增强的主要原因：R211 L，A217 V，E356 K和S364 C。因此，用非致死菌落筛选体内生物发光活性的定向进化被证明是增加荧光素酶的热稳定性同时保持高催化活性的有效和高效的方法。
Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42 degrees C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K-m for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.