New fast BiFC plasmid assay system for in vivo protein-protein interactions.
In this age of massive genetic and protein information, a fast and reliable method of studying in vivo protein-protein interactions is necessary. We have developed a novel system that can overcome limitations of existing assay methods. This new method adopts two existing systems for fast analysis of diverse protein-protein interactions. For rapid, large-scale cloning, we adopted the Gateway system and developed novel destination vectors containing YFP N-terminus (YN) or YFP C-terminus (YC) to visualize protein-protein interactions in vivo using bimolecular fluorescence complementation (BiFC). Using this system, we investigated molecular interactions among the three POZ-domain regulatory proteins mAPM-1, LRF, KLHL10 that belong to a subgroup of human POZ-domain proteins, and showed that the POZ-domains of mAPM-1, LRF and KLHL10 could form both homodimers and heterodimers. This new method is a highly efficient, sensitive and specific assay method for protein-protein interaction in vivo.
Kim
Myung-Hwa
MH
Department of Biochemistry and Molecular Biology, BK21 Project for Medical Science, Institute of Genetic Science, Yonsei University School of Medicine, Seoul (Korea).
Roh
Hee-Eun
HE
Lee
Min-Nyung
MN
Hur
Man-Wook
MW
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D011506:Proteins; D005982:Glutathione Transferase