Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

Abstract

We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting D-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.Luciferases are enzymes that emit light in reaction with a specific substrate in the presence of cofactors. A diverse group of organisms use luciferase-mediated bioluminescence to startle predators or to attract prey or mates. The emitted light is used as a detection system for luciferase expression, which acts as a "reporter" for the activity of any regulatory elements that control its expression. Luciferase is particularly useful as a reporter enzyme in living cells and organisms. Firefly luciferase is one among many of the sensitive luciferases and is widely used by researchers to identify different biological events of cells in culture and in living small animals, and it is also used by public health researchers for the detection of food contamination. [1][2][3] The gene encoding firefly luciferase, cloned in 1985 from the North American firefly, Photinus pyralis, is now emerging as the gene of choice for in vivo and in vitro reporting of transcriptional activity in eukaryotic cells. 4 Reporter genes encoding for proteins with optical signatures, either fluorescent or bioluminescent, are a low-cost alternative for realtime analysis of gene expression in small-animal ...

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