Labeling Ebola Virus with a Self-Splicing Fluorescent Reporter.
Labeling Ebola Virus with a Self-Splicing Fluorescent Reporter.
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DOI：
10.3390/microorganisms10112110
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发表时间：
2022-10-26
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
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内含肽（插入蛋白质）是一种多肽，它可以中断其他蛋白质的序列，并通过蛋白质剪接去除自身。在这种内含肽催化的反应中，内含肽周围的两个肽键被重排，以从侧翼蛋白质序列释放内含肽，称为N-和C-外显肽，它们同时通过肽键连接。由于这种独特的功能，内含肽已被证明在蛋白质工程中非常有用。先前的工作已经证明，异源蛋白质可以插入内含肽内，内含肽和插入的蛋白质都保留功能，允许含有内含肽的基因共表达额外的编码序列。在这里，我们表明，荧光蛋白（ZsGreen）可以插入Pyrococcus horikoshii RadA内含肽，与杂交蛋白（ZsG-Int）保持荧光和剪接能力。我们使用这个系统来创建表达荧光蛋白的重组埃博拉病毒。我们首先测试了单个埃博拉病毒蛋白中ZsG-Int的多个潜在插入位点，确定了VP 30基因中的一个位点，该位点有助于哺乳动物细胞中有效的内含肽剪接，同时也保留了VP 30的功能。接下来，我们成功地拯救了含有ZsG-Int-VP 30融合蛋白的病毒，该融合蛋白在感染的细胞中显示荧光。因此，我们报告了一个新的内含肽为基础的应用程序添加报告系统，而不需要添加额外的基因。此外，这项工作突出了一种新的报告基因设计，其中报告基因仅在感兴趣的蛋白质被翻译并且不保持与感兴趣的蛋白质融合的情况下产生。
Inteins (intervening proteins) are polypeptides that interrupt the sequence of other proteins and remove themselves through protein splicing. In this intein-catalyzed reaction, the two peptide bonds surrounding the intein are rearranged to release the intein from the flanking protein sequences, termed N- and C-exteins, which are concurrently joined by a peptide bond. Because of this unique functionality, inteins have proven exceptionally useful in protein engineering. Previous work has demonstrated that heterologous proteins can be inserted within an intein, with both the intein and inserted protein retaining function, allowing for intein-containing genes to coexpress additional coding sequences. Here, we show that a fluorescent protein (ZsGreen) can be inserted within the Pyrococcus horikoshii RadA intein, with the hybrid protein (ZsG-Int) maintaining fluorescence and splicing capability. We used this system to create a recombinant Ebola virus expressing a fluorescent protein. We first tested multiple potential insertion sites for ZsG-Int within individual Ebola virus proteins, identifying a site within the VP30 gene that facilitated efficient intein splicing in mammalian cells while also preserving VP30 function. Next, we successfully rescued a virus containing the ZsG-Int-VP30 fusion protein, which displayed fluorescence in the infected cells. We thus report a new intein-based application for adding reporters to systems without the need to add additional genes. Further, this work highlights a novel reporter design, whereby the reporter is only made if the protein of interest is translated and does not remain fused to the protein of interest.
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