A single-chain antibody/epitope system for functional analysis of protein-protein interactions.
Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.
Fujiwara
Kosaku
K
Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3301, USA.
Poikonen
Kari
K
Aleman
Lourdes
L
Valtavaara
Minna
M
Saksela
Kalle
K
Mayer
Bruce J
BJ
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D050737:Proto-Oncogene Proteins c-crk; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C493872:Crk protein, rat; D000939:Epitopes; D007135:Immunoglobulin Variable Region; C078332:Nck protein; D015513:Oncogene Proteins; D019151:Peptide Library; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D016271:Proto-Oncogene Proteins c-myc; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl