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

Escherichia coli is one of the most widely used vehicles to overexpress mem brane proteins (MPs). Currently, it is not possible to predict if an overex pressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to o verexpression in inclusion bodies, since it is relatively easy to isolate M Ps, from membranes and usually impossible to isolate them from inclusion bo dies. Here we show that green fluorescent protein (GFP), when fused to an o verexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermor e, we show that an overexpressed MP can be recovered from a MP-GFP fusion u sing a site specific protease. This makes GFP an excellent toot for large-s cale MP target selection in structural genomics projects. (C) 2001 Federati on of European Biochemical Societies. Published by Elsevier Science B.V. Al l rights reserved.