Use of GFP tags to monitor localization of different luciferases in E. coli.
The utility of the green fluorescent protein (GFP) as a probe to monitor protein localization in living cells is gaining a great deal of attention. In this study, to understand the localization of luciferases in E. coli, we have attached GFP tags at both the N- and the C-terminus of firefly luciferase (FF-Luc)(from Pyrocoelia miyako) and of red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), respectively. There was no significant change in the bioluminescence emission spectrum for any of the three luciferases following the tagging with GFP at either the N- or C-terminus, confirming the absence of energy transfer between one another. Using confocal imaging microscopy, we observed that all three luciferases expressed in the E.coli cultured at 37 degrees C tend to aggregate and are seen to localize in the poles, thus confirming their poor folding properties. In contrast, in the E.coli cultured at 18 degrees C FF-Luc was found to be highly expressed in the soluble form when compared to RE-Luc and GR-Luc. These results support our previous finding that the folding properties of FF-Luc and RE/GR-Luc are totally different.
Venkatesh
Balan
B
Cell Dynamics Research Group, National Institute of Advanced Industrial Science and Technology, Midorigaoka Ikeda, Osaka 563-8577, Japan.
Arifuzzaman
Mohammad
M
Mori
Hirotada
H
Suzuki
Shingo
S
Taguchi
Takashisa
T
Ohmiya
Yoshihiro
Y
D003600:Cytosol; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006358:Hot Temperature; D008156:Luciferases / Q000378:metabolism*; D011489:Protein Denaturation
D049452:Green Fluorescent Proteins; D008156:Luciferases