White P J, Squirrell D J, Arnaud P, Lowe C R, Murray J A
Institute of Biotechnology, University of Cambridge, U.K.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):343-50. doi: 10.1042/bj3190343.
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.
我们利用随机化学诱变和简单的遗传筛选方法，从北美萤火虫（Photinus pyralis）中生成并分离出一种热稳定的荧光素酶突变体。单个G到A的转换突变，导致蛋白质序列中第354位的赖氨酸残基被谷氨酸取代，已证明这种突变是这种增强的热稳定性的原因。使用定点诱变实现了用所有可能的氨基酸残基替换Glu-354，并产生了一系列具有不同热稳定性的突变酶。突变E354K和E354R使热稳定性增加最多，这表明侧链大小、疏水性以及电荷，可能也是该位置多肽链整体热稳定性的重要贡献因素。对于此类突变而言不同寻常的是，生化研究表明该位置位于蛋白质表面并暴露于溶剂中。
