一种基因编码的细胞内铜离子荧光探针
首发时间:2012-05-11
摘要:作为生物体内最重要的金属元素之一,铜通过辅因子的形式广泛参与生命过程,与蛋白质、核酸等大分子相互作用,在生命医学科学中扮演了重要角色。生物体对于细胞内的铜离子含量有严格的调控机制,铜的过剩或缺乏都会导致疾病。因此,针对铜离子进行定性或定量的特异性检测对生命医学科学具有重要意义,能够实时动态观察活细胞内铜离子状态的方法逐渐成为研究的热点。本文利用原核细胞中的转录调控蛋白Fur对金属选择性结合的特点,融合荧光蛋白突变体,利用基因工程的方法构建了一系列铜离子荧光蛋白探针样品,通过活菌检测筛选获得了对铜离子响应高选择性好的荧光探针FS-4,可直接用于细胞内的铜离子实时动态检测。
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A Genetically Encoded Fluorescent Biosensor for Copper in Living Cells
Abstract:Copper is one of the most important element for life, which is required mainly in the form of cofactor for biological processes. Organisms developed sophisticated mechanism for metabolism of this metal, which has been kept in a narrow range in cell, because misregulation of copper is known to result in neurodegeneration diseases. Therefore, a real-time detection method for visualize copper inside is significant for farther research. We developed a genetically encoded fluorescent copper sensor basing on cpYFP and a responsor of gene transcription Fur. With protein engineering and molecular biology technology, we construct a series of fusion protein varieties as sensor candidates. The final biosensor FS-4, which is achieved by whole-cell screening, performed dramatically fluorescent changes towards copper binding. With high sensitivity and selectivity, FS-4 will be used for copper detection in living cells.
Keywords: fluorescent protein copper ion sensor Fur
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