Development of a destabilized firefly luciferase enzyme for measurement ofgene expression

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 e nabled multiple measurements of gene expression in the same living cell. Al though this approach has already provided new insights about expression dyn amics, its future utility is limited by the three- to four-hour half-life o f firefly luciferase in mammalian cells. Because of this, rapid increases o r decreases in gene expression may not be detected, owing to the accumulati on of residual luciferase. Accordingly, the goal of the present study was t o develop a luciferase reporter with a reduced functional half-life. This w as accomplished by adding a synthetic fragment to the firefly luciferase-co ding sequence that encoded the proteolytic "PEST" signal from mouse ornithi ne decarboxylase. When placed under the control of estrogen response elemen ts and expressed in human breast cancer T-47D cells, the modified luciferas e protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared t o 3.68 h for the wild-type enzyme. As anticipated the overall rate of photo nic emissions in cells expressing the destabilized luciferase was about sev enfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the phot onic activity derived from LUCODC-DA was still sufficient to enable real-ti me measurements of gene expression in single living cells.