[Assessment of the Escherichia coli Tat protein translocation system with fluorescent proteins].
The possibility of using fluorescent proteins as probes to study the twin-arginine translocation (Tat) system was assessed in Escherichia coli. When fused to the twin-arginine signal peptide of trimethylamine N-oxide reductase, the DsRed2 red fluorescent protein from the Discosoma sp. was successfully synthesized and folded in E. coli cells. However, RR-DsRed2 aggregated inside the cells. Therefore, although DsRed2 has been engineered from DsRed for faster maturation and lower non-specific aggregation, it is still not compatible with Tat-dependent translocation. In contrast, the jellyfish green fluorescent protein (GFP) was efficiently exported into periplasm even when the RR motif was changed to KR or RK. These results show that GFP can be used as an efficient reporter protein to study Tat system, but DsRed2 is not suitable for such purpose because of its aggregation property. In addition, when the protein concentration was similar, the fluorescence intensity of KR-GFP and RK-GFP decreased compared with RR-GFP, which would suggest that the twin-arginine signal peptide is not only essential for mediating protein translocation, but also important for the folding of down-stream protein.
Zhang
Ming
M
Key Lab of Ion Beam Bioengineering, Institute of Plasma Physics, the Chinese Academy of Sciences, Hefei, China.
Pan
Ren-Rui
RR
Yu
Zeng-Liang
ZL
Wu
Long-Fei
LF
D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000502:physiology*; D010089:Oxidoreductases, N-Demethylating / Q000502:physiology; D017510:Protein Folding; D021382:Protein Sorting Signals
D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C473116:TatB protein, E coli; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase