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首页> 外文期刊>Journal of Biotechnology >Fluorescence complementation via EF-hand interactions
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Fluorescence complementation via EF-hand interactions

机译：通过EF-手相互作用的荧光互补

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Fluorescence complementation technology with fluorescent proteins is a powerful approach to investigate molecular recognition by monitoring fluorescence enhancement when non-fluorescent fragments of fluorescent proteins are fused with target proteins, resulting in a new fluorescent complex. Extension of the technology to calcium-dependent protein-protein interactions has, however, rarely been reported. Here, a linker containing trypsin cleavage sites was grafted onto enhanced green fluorescent protein (EGFP). Under physiological conditions, a modified fluorescent protein, EGFP-T1, was cleaved into two major fragments which continue to interact with each other, exhibiting strong optical and fluorescence signals. The larger fragment, comprised of amino acids 1-172, including the chromophore, retains only weak fluorescence. Strong green fluorescence was observed when plasmid DNA encoding complementary EGFP fragments fused to the EF-hand motifs of calbindin D9k (EF1 and EF2) were co-transfected into HeLa cells, suggesting that chromophore maturation and fluorescence complementation from EGFP fragments can be accomplished intracellularly by reassembly of EF-hand motifs, which have a strong tendency for dimerization. Moreover, an intracellular calcium increase upon addition of a calcium ionophore, ionomycin in living cells, results in an increase of fluorescence signal. This novel application of calcium-dependent fluorescence complementation has the potential to monitor protein-protein interactions triggered by calcium signalling pathways in living cells.
机译：荧光蛋白的荧光互补技术是一种强大的方法，可通过在荧光蛋白的非荧光片段与目标蛋白融合形成新的荧光复合物时监测荧光增强来研究分子识别。然而，很少有报道将该技术扩展到钙依赖性蛋白-蛋白相互作用。在这里，包含胰蛋白酶切割位点的接头被嫁接到增强的绿色荧光蛋白（EGFP）上。在生理条件下，将修饰的荧光蛋白EGFP-T1切割成两个主要片段，它们继续彼此相互作用，表现出较强的光学和荧光信号。由氨基酸1-172组成的较大片段（包括发色团）仅保留弱荧光。当将编码与calbindin D9k的EF手基序融合的互补EGFP片段（EF1和EF2）的质粒DNA共转染HeLa细胞时，观察到强烈的绿色荧光，这表明通过EGFP片段的发色团成熟和荧光互补可以通过在细胞内完成EF手形图案的重新组装，这些图案具有很强的二聚化趋势。此外，在活细胞中添加钙离子载体离子霉素后，细胞内钙增加，导致荧光信号增加。钙依赖性荧光互补的这一新应用具有监测活细胞中钙信号通路触发的蛋白质-蛋白质相互作用的潜力。

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