Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Photochem Photobiol Sci. 2011 Jul;10(7):1203-15. doi: 10.1039/c1pp05012e. Epub 2011 Apr 15.
The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make three separate mutant enzymes with a single bridge. Moreover, C(81)-A(105)C mutant luciferase was modified and successfully secreted to the extracellular medium. By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red and the optimum temperature of activity was also increased (up to 10 °C more than wild type). Amongst mutants with a disulfide bridge, P(451)C-V(469)C and L(306)C-L(309)C mutants exhibit a single peak in the red region of the spectrum at pH 7.8. It is worthwhile to note that with the design of a secreted luciferase, the increased optimum temperature, thermostability and emission of red light might make mutant luciferase suitable reporters for the study of gene expression in high through-put screening.
生物发光反应利用荧光素、Mg(2+)-ATP 和分子氧产生电子激发态氧荧光素，由荧光素酶进行，并发出可见光。萤火虫荧光素酶的生物发光颜色取决于荧光素酶结构和测定条件。有人提出，通过引入二硫键可以增加蛋白质的稳定性，从而降低展开的构象熵。本研究将二硫键引入 Photinus pyralis 荧光素酶中，得到了带有单个桥的三个独立突变酶。此外，还对 C(81)-A(105)C 突变体荧光素酶进行了修饰，并成功分泌到细胞外培养基中。通过在 Photinus pyralis 荧光素酶中进行定点突变引入二硫键，发光颜色变为红色，并且活性的最适温度也提高了（比野生型高 10°C 左右）。在带有二硫键的突变体中，P(451)C-V(469)C 和 L(306)C-L(309)C 突变体在 pH 7.8 时在光谱的红色区域显示出单个峰。值得注意的是，通过设计分泌型荧光素酶，最适温度、热稳定性和红光的增加可能使突变荧光素酶适合用于高通量筛选中基因表达的研究。
