Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing pr oteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed th e mechanism and capabilities of the E. coli Tat system using green fluoresc ent protein (GFP) fused to the twin-arginine signal peptide of TMAO reducta se (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is a lmost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type ce lls corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work h as shown GFP to be incapable of folding in the periplasm, we propose that G FP is exported in a fully folded, active state. These data also show for th e first time that heterologous proteins can be exported in an active form b y the Tat pathway.