A Single-Chain Antibody/Epitope System for Functional Analysis of Protein−Protein Interactions†

ABSTRACT : 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 iscurrently 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 consequencesto be determined. For this purpose, we have developed an artificial binding interface consisting of aanti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFvderived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improvedclone, 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. Invivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improvedby including multiple tandem copies of the Myc epitope in the binding partner. To test the system, wesuccessfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptorproteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expectthat this approach, which we term a functional interaction trap, will be a powerful proteomic tool forinvestigating protein-protein interactions.In cellular signal transduction, protein-protein interactionsplay a critical role in regulating processes such as prolifera-tion, adhesion, and differentiation. Signaling proteins oftencontain one or more modular protein-protein bindingdomains such as Src homology 2 (SH2)