A time- and cost-efficient system for high-level protein production in mammalian cells
A time- and cost-efficient system for high-level protein production in mammalian cells
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DOI：
10.1107/s0907444906029799
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发表时间：
2006-10-01
影响因子：
2.2
通讯作者：
Jones, E. Yvonne
中科院分区：
文献类型：
--
作者：
Aricescu, A. Radu;Lu, Weixian;Jones, E. Yvonne
大多数用于结构生物学研究的蛋白质是通过在大肠杆菌中高水平表达产生的。然而，基于原核的表达系统不能产生许多蛋白质的正确折叠的功能形式，因此已经开发了多种基于真核的表达系统。其中，酵母和杆状病毒感染的昆虫细胞目前代表了结构生物学家选择的表达系统。在这里，一个简单，快速和负担得起的方法在哺乳动物细胞中的瞬时蛋白质表达的协议。结果表明，它结合了生产用于结构生物学，特别是蛋白质晶体学的合适样品所必需的几个特征，即高蛋白质产率、直接纯化、硒代甲硫氨酸掺入和N-连接的糖基化的控制。该系统适用于常规实验室，也可以在中等或高通量管道中实施。
Most proteins for structural biology studies are produced by high- level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus- infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N- linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.