Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
Antioxid Redox Signal. 2011 Mar 15;14(6):973-84. doi: 10.1089/ars.2010.3712. Epub 2011 Jan 23.
The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia coli. This trypsin inhibitor contains three disulfides that contribute to its extreme stability and protease resistance. The mutants we isolated for increased expression appear to act by eliminating or destabilizing the Cys14-Cys38 disulfide in BPTI. In doing so, they are expected to reduce or eliminate kinetic traps that exist within the well characterized in vitro folding pathway of BPTI. These results suggest that elimination or destabilization of a disulfide bond whose formation is problematic in vitro can enhance in vivo protein folding. The use of these in vivo selections may prove a valuable way to identify and eliminate disulfides and other rate-limiting steps in the folding of proteins, including those proteins whose in vitro folding pathways are unknown.
周质空间提供了一个强氧化的环境；然而，具有二硫键的周质空间表达蛋白质的效率通常不高。在这里，我们使用两种不同的三联体融合系统，对模型蛋白牛胰蛋白酶抑制剂（BPTI）的突变体进行体内选择，目的是提高其在大肠杆菌中的表达。这种胰蛋白酶抑制剂含有三个二硫键，有助于其极端稳定性和抗蛋白酶性。我们分离出的用于提高表达的突变体似乎通过消除或破坏 BPTI 中 Cys14-Cys38 二硫键来发挥作用。这样做，它们有望减少或消除 BPTI 体内折叠途径中存在的动力学陷阱。这些结果表明，消除或破坏在体外形成有问题的二硫键可以增强体内蛋白质折叠。这些体内选择的使用可能被证明是一种有价值的方法，可以识别和消除蛋白质折叠中的二硫键和其他限速步骤，包括那些其体外折叠途径未知的蛋白质。
