Firefly luciferase enzyme fragment complementation for imaging in cells and living animals
Firefly luciferase enzyme fragment complementation for imaging in cells and living animals
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DOI：
10.1021/ac0484777
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发表时间：
2005-03-01
影响因子：
7.4
通讯作者：
Gambhir, SS
中科院分区：
文献类型：
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作者：
Paulmurugan, R;Gambhir, SS
我们根据萤火虫荧光素酶的晶体结构，鉴定出萤火虫荧光素酶基因的不同片段。与目前用于研究蛋白质相互作用的片段不同，这些编码蛋白质片段的分裂报告基因可以在培养和活体小鼠的不同细胞系中自我补充并提供荧光素酶活性。对不同片段组合的萤火虫荧光素酶活性的片段互补相关恢复与完整萤火虫荧光素酶活性的比较进行了估计，其范围从0.01到4%不等。使用冷却的光学电荷耦合器件相机，分析瞬时转染的活小鼠皮下293T细胞植入物中萤火虫荧光素酶片段的互补，发现注射d -荧光素后可检测到显著的酶活性，特别是从鉴定片段的组合（Nfluc和Cfluc分别是萤火虫荧光素酶基因的N和C片段）：Nfluc (1-475)/Cfluc（245-550）、Nfluc (1-475)/Cfluc（265-550）和Nfluc (1-475)/Cfluc（300-550）。与SV40的核定位信号（NLS）肽一起表达的Cfluc（265-550）片段与不含NLS表达的Nfluc（1-475）片段共转染后，酶活性降低。通过将TAT-Cfluc（265-550）传递到稳定表达Nfluc（1-475）的细胞中并恢复信号，我们在本研究中也证明了互补片段可以有效地用于筛选大分子递送载体。这些互补片段应该对许多基于报告的分析有用，包括细胞内蛋白质定位，研究细胞大分子递送载体，研究细胞-细胞融合，以及基于片段互补的细胞内磷酸化传感器。
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 TAT-Cfluc (265-550) 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.