Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.
BACKGROUND
Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.


RESULTS
Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.


CONCLUSIONS
The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.
Kobayashi
Takuya
T
Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.
Morone
Nobuhiro
N
Kashiyama
Taku
T
Oyamada
Hideto
H
Kurebayashi
Nagomi
N
Murayama
Takashi
T
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003599:Cytoskeleton / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary
D049452:Green Fluorescent Proteins; D006639:Histidine