Development of a destabilized firefly luciferase enzyme for measurement of gene expression
Development of a destabilized firefly luciferase enzyme for measurement of gene expression
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DOI：
10.2144/00293rr02
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发表时间：
2000-09-01
期刊：
影响因子：
2.7
通讯作者：
Frawley, LS
中科院分区：
文献类型：
--
作者：
Leclerc, GM;Boockfor, FR;Frawley, LS
萤火虫荧光素酶被广泛用作基因调节和表达研究的报告酶。结合荧光素酶报告基技术和数字成像显微镜的新技术的最新发展使得在同一活细胞中的基因表达进行了多次测量。尽管这种方法已经提供了有关表达动力学的新见解，但其未来效用受哺乳动物细胞中萤火虫荧光素酶的三个小时半衰期的限制。因此，由于残留荧光素酶的积累，可能无法检测到基因表达的快速增加或降低。因此，本研究的目的是开发荧光素酶记者，其功能性半衰期降低。这是通过在萤火虫荧光素酶编码序列中添加合成片段来完成的，该序列编码了小鼠鸟氨酸脱羧酶的蛋白水解“害虫”信号。当置于雌激素反应元件的控制下并在人类乳腺癌T-47D细胞中表达时，修饰的荧光素酶蛋白（LucoDC-DA）的功能性半衰期为0.84 h，而野生型酶为3.68 h。正如预期的那样，表达不稳定的荧光素酶的细胞中光子排放的总速率比其野生型对应物低约七倍，这大概是因为稳态荧光素酶的积累减少了。即便如此，源自LucoDC-DA的光子活性仍然足以实现单个活细胞中基因表达的实时测量。
Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.