Lee, You Ri Park, Jong-Hwa Hahm, Soo-Hyun Kang, Lin-Woo Chung, Ji Hyung Nam, Ki-Hyun Hwang, Kwang Yeon Kwon, Ick Chan Han, Ye Sun 2024-01-20T18:32:43Z 2024-01-20T18:32:43Z 2021-09-04 2010-10 1536-1632 https://pubs.kist.re.kr/handle/201004/131080 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. 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)x2] 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. 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. 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. English SPRINGER HUMAN CHECKPOINT SENSOR BASE EXCISION-REPAIR DNA-POLYMERASE-BETA HUMAN MUTY HOMOLOG RAD9-RAD1-HUS1 INTERACTS IN-VIVO CLAMP COMPLEX SUBSTRATE ENZYME Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein-Protein Interactions in Cells Article 10.1007/s11307-010-0312-2 1 MOLECULAR IMAGING AND BIOLOGY, v.12, no.5, pp.468 - 478 MOLECULAR IMAGING AND BIOLOGY 12 5 468 478 scie scopus 000282273200003 2-s2.0-78049348351 Radiology, Nuclear Medicine & Medical Imaging Radiology, Nuclear Medicine & Medical Imaging Article HUMAN CHECKPOINT SENSOR BASE EXCISION-REPAIR DNA-POLYMERASE-BETA HUMAN MUTY HOMOLOG RAD9-RAD1-HUS1 INTERACTS IN-VIVO CLAMP COMPLEX SUBSTRATE ENZYME Bimolecular fluorescence complementation Dronpa Reversible photoswitching activity Protein-protein interaction Human MutY homolog hHus1 hRad1