Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli.
The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.
Barrett
Claire M L
CM
Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK.
Ray
Nicola
N
Thomas
Joanne D
JD
Robinson
Colin
C
Bolhuis
Albert
A
D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase