TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector
TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector
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DOI：
10.1002/pro.518
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发表时间：
2010-12-01
期刊：
影响因子：
8
通讯作者：
Truong, Kevin
中科院分区：
文献类型：
--
作者：
Chen, Xi;Pham, Elizabeth;Truong, Kevin
多个基因的递送和表达是一系列应用中的重要要求，例如合成信号传导途径的工程化和多能干细胞的诱导。然而，常规方法通常效率低下，我们在此描述了一种使用单一表达载体通过模拟非化学计量并且可能限制可同时表达的基因的最大数目的多基因递送的通用方法。RNA病毒的蛋白质表达策略这是通过首先将基因与TEV蛋白酶一起表达为单一融合蛋白，然后将融合蛋白进行蛋白水解自切割成功能组分来实现的。为了在大肠杆菌细胞中证明该方法，我们使用SDS-PAGE分析了翻译产物，并显示融合蛋白被有效地切割成其组分，然后可以将其单独纯化或作为结合复合物纯化。为了在哺乳动物细胞中证明该方法，我们设计了一种差异定位方案，并使用活细胞成像来观察加工产物的独特亚细胞靶向。我们还表明，加工产物的化学计量是一致的，并与加工产物的频率相对应。总之，使用单一TEV蛋白酶自加工载体在大肠杆菌和哺乳动物细胞中实现了多达三种基因的有效表达和分离
Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components To demonstrate this method in E colt cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector In summary, the efficient expression and separation of up to three genes was achieved in both E coli and mammalian cells using a single TEV protease self-processing vector