A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.
A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.
复制标题
能够增强哺乳动物细胞中 RNA 表达和蛋白质合成的前导序列。
DOI：
10.1002/pro.2325
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Chaput,JohnC
中科院分区：
文献类型：
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作者：
Wellensiek,BrianP;Larsen,AndrewC;Flores,Julia;Jacobs,BertramL;Chaput,JohnC
生物技术中的许多应用需要从人类细胞产生的人类蛋白质。通常用于此目的的稳定细胞系难以开发，并且扩展到大量蛋白质可能是有问题的。瞬时表达可以避免这个问题，但蛋白质产量通常太低，大多数应用。在这里，我们报告了一种新的37个核苷酸的前导序列，可促进哺乳动物细胞中快速和高转基因表达。该序列通过体外选择鉴定，并在基于牛痘的瞬时细胞质表达系统中发挥作用。含有该序列的载体在106个细胞中的小规模表达中仅在6小时内产生微克水平的蛋白质。这种水平的蛋白质合成对于高通量生产人类蛋白质是理想的，并且可以按比例产生毫克量的蛋白质。该技术与广泛的细胞系兼容，接受质粒和线性DNA，并与批准在BSL 1条件下使用的病毒一起发挥作用。我们认为，这些优点提供了一个强大的方法，在哺乳动物细胞中产生人类蛋白质。
Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37‐nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified byin vitroselection and functions in a transient vaccinia‐based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small‐scale expression in 106cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.