Kyratsous Christos A, Panagiotidis Christos A
Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
Methods Mol Biol. 2012;824:109-29. doi: 10.1007/978-1-61779-433-9_5.
Molecular chaperones or heat-shock proteins (HSPs) are protein machines that interact with unfolded or partially folded polypeptides and assist them in attaining their proper conformation. The folding reaction relies on a complex array of scaffolding effects and ATP-driven conformational changes that mediate the temporary unfolding and subsequent refolding of protein substrates. DnaK and GroEL are the two major Escherichia coli chaperones. They belong to the HSP70 and HSP60 families of proteins, respectively, and play a major role in protein folding. Here, we describe a set of bacterial expression vectors that permits the fusion of a protein of interest to DnaK or GroEL and its subsequent quantitative expression in a soluble, easily purifiable form. We also provide a set of compatible co-chaperone expression constructs that permit the simultaneous co-expression of the DnaK and GroEL physiological partners to further increase protein solubility. The system was successfully tested using the murine prion protein (PrP). Although PrP is normally insoluble when expressed in E. coli, we show that utilizing our vectors it can be produced in a soluble form as a DnaK or GroEL fusion. This system is useful for the production of a large array of proteins that fail to fold properly when expressed in E. coli.
分子伴侣或热休克蛋白（HSPs）是与未折叠或部分折叠的多肽相互作用并协助其获得正确构象的蛋白质机器。折叠反应依赖于一系列复杂的支架效应和ATP驱动的构象变化，这些变化介导蛋白质底物的暂时展开和随后的重新折叠。DnaK和GroEL是大肠杆菌的两种主要伴侣蛋白。它们分别属于HSP70和HSP60蛋白家族，在蛋白质折叠中起主要作用。在这里，我们描述了一组细菌表达载体，这些载体允许将感兴趣的蛋白质与DnaK或GroEL融合，并随后以可溶性、易于纯化的形式进行定量表达。我们还提供了一组兼容的共伴侣表达构建体，允许同时共表达DnaK和GroEL的生理伴侣，以进一步提高蛋白质的溶解度。该系统已使用小鼠朊病毒蛋白（PrP）成功进行了测试。尽管PrP在大肠杆菌中表达时通常不溶，但我们表明利用我们的载体它可以作为DnaK或GroEL融合蛋白以可溶形式产生。该系统对于生产在大肠杆菌中表达时无法正确折叠的大量蛋白质很有用。
