Drew D E, von Heijne G, Nordlund P, de Gier J W
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden.
FEBS Lett. 2001 Oct 26;507(2):220-4. doi: 10.1016/s0014-5793(01)02980-5.
Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.
大肠杆菌是用于过表达膜蛋白（MPs）的最广泛使用的载体之一。目前，无法预测过表达的MP最终会存在于细胞质膜中还是包涵体中。与在包涵体中过表达相比，在细胞质膜中过表达MP受到强烈青睐，因为从膜中分离MP相对容易，而从包涵体中分离通常是不可能的。在这里，我们表明，绿色荧光蛋白（GFP）与过表达的MP融合时，可以用作监测大肠杆菌中过表达的MP的膜插入与包涵体形成的指标。此外，我们表明可以使用位点特异性蛋白酶从MP-GFP融合物中回收过表达的MP。这使得GFP成为结构基因组学项目中大规模MP靶点选择的优秀工具。
