Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli

FEBS Letters

Nordlund, Pär¹  von Heijne, Gunnar¹  Drew, David E¹  de Gier, Jan-Willem L¹ 

[1] Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden

关键词: Membrane protein;    Green fluorescent protein;    Protein overexpression;    Escherichia coli;    MP;    membrane protein;    GFP;    green fluorescent protein;    GST;    glutathione-S-transferase;    TEV;    tobacco etch virus;   

DOI  :  10.1016/S0014-5793(01)02980-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown