Improved thermostability of the North American firefly luciferase: Saturation mutagenesis at position 354
Improved thermostability of the North American firefly luciferase: Saturation mutagenesis at position 354
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DOI：
10.1042/bj3190343
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发表时间：
1996-10-15
影响因子：
4.1
通讯作者：
Murray, JAH
中科院分区：
文献类型：
--
作者：
White, PJ;Squirrell, DJ;Murray, JAH
我们利用随机化学诱变和简单的遗传筛选，从北美萤火虫(Photinus Pyralis)中产生并分离到一个耐热的荧光素酶突变体。一个单一的G-A转换突变，导致蛋白质序列中第354位的谷氨酸取代赖氨酸残基，被证明是导致这种热稳定性增强的原因。用所有可能的氨基酸残基替换Glu-354是通过定向突变实现的，并产生了具有一系列热稳定性的突变酶。突变E354K和E354R在热稳定性方面的提高最大，这表明侧链的大小和疏水性以及电荷也可能是影响该位置多肽链整体热稳定性的重要因素，对于这种突变，生化研究表明该位置位于蛋白质的表面并暴露在溶剂中。
We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position, Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.