利用荧光蛋白对大肠杆菌蛋白质Tat转运系统的研究

探讨了荧光蛋白作为报告蛋白用于蛋白质转运系统研究的可行性 ,结果表明海葵红色荧光蛋白聚集在细胞质内 ,不能转运至周质空间。而水母绿色荧光蛋白在Tat信号肽和Tat转运酶的共同作用下 ,以折叠形式转运至周质空间。通过荧光定量分析表明信号肽保守序列中的双精氨酸是保证绿色荧光蛋白转运及转运效率所必需的 ,且第二个精氨酸比第一个精氨酸更为重要。同时 ,揭示了Tat信号肽需要一定的高级结构才能行使功能 ;Tat信号肽不仅引导蛋白质的转运 ,而且也参与蛋白质的折叠。因此 ,绿色荧光蛋白是非常理想的报告蛋白 ,可用于研究Tat系统 ,但是海葵红色荧光蛋白易于聚集而不适合于此目的。

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.