title: Tracking protein-protein interaction and localization in living cells using a high-affinity molecular binder.
abstract: Probing protein-protein interactions in living cells is crucial for understanding the protein functions and developing drugs. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time through live-cell imaging. A repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. The potential and utility of our approach was demonstrated by tracking the rapamycin-mediated interaction between FKBP12-rapamycin binding (FRB) domain and a FK506-binding protein (FKBP) and their localization by live-cell imaging. The present approach can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells.
authors.affiliation: Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
authors.forename: Hee-Yeon
authors.initials: HY
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
authors.forename: Joong-jae
authors.initials: JJ
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
authors.forename: Nury
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
authors.forename: Won Do
authors.initials: WD
authors.lastname: Heo
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea. Electronic address: hskim76@kaist.ac.kr.
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
mesh_terms: D008164:Luminescent Proteins / Q000378:metabolism*; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000378:metabolism*; D016559:Tacrolimus / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D015335:Molecular Probes; D016559:Tacrolimus; D022061:Tacrolimus Binding Protein 1A; D000097573:Red Fluorescent Protein
keywords: Colocalization analysis; Intracellular antibody; Live-cell imaging; Protein–protein interactions; Red fluorescent protein