Development of bimolecular fluorescence complementation using Dronpa for visualization of protein-protein interactions in cells.
OBJECTIVE
We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein-protein interactions in cells.


METHODS
Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1-Glu164), Dronpa C-terminal: DC (Gly165-Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)×2] was introduced to enhance Dronpa complementation by hHus1-hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region.


RESULTS
Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1-LDN and DCL-hRad1 expression vectors, but not with hHus1-LDN or DCL-hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH-LDN and DCL-hHus1 expression vectors or hMYH-LDN and hHus1-LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH-LDN and DCL-hHus1, showed almost identical photoswitching activity as that of native Dronpa.


CONCLUSIONS
These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein-protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein-protein interaction.
Lee
You Ri
YR
Department of Advanced Technology Fusion, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea.
Park
Jong-Hwa
JH
Hahm
Soo-Hyun
SH
Kang
Lin-Woo
LW
Chung
Ji Hyung
JH
Nam
Ki-Hyun
KH
Hwang
Kwang Yeon
KY
Kwon
Ick Chan
IC
Han
Ye Sun
YS
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D011506:Proteins