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 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.
Drew
D E
DE
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden.
von Heijne
G
G
Nordlund
P
P
de Gier
J W
JW
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; C064459:KDEL receptor; D008164:Luminescent Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C490210:ProW protein, E coli; D011498:Protein Precursors; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C412443:YIDC protein, E coli; C025668:coat protein, Bacteriophage M13; D049452:Green Fluorescent Proteins