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 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.

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions

“…Although immunoprecipitation has been used extensively and traditionally for obtaining quantitative kinetic and binding information, quantification is a laborious process, as well the instrumentation is large and not readily available [15]. In contrast, the yeast two-hybrid system and phage display technologies cannot be used for kinetic information, as well the techniques can be complex, restricted to certain systems and time-consuming [12,16,17]. Similarly, peptide and protein domain arrays can be used for qualitative analyses, but these methods have not been reduced to practicality and they are not quantitative [18].…”

Functional Interaction Trap

“…Modifications-FIT was conceived as a general strategy to probe the functional consequences of interaction between two proteins (17). Here we show that this approach can be adapted to induce the efficient tyrosine phosphorylation in the cell of a single protein of choice, in the absence of significantly increased phosphorylation of other proteins.…”

“…Substrate Constructs-For each substrate, PCR was used to generate full-length constructs with 5Ј BamHI and 3Ј NotI sites (17). These fragments were ligated into the BamHI and NotI sites of pEBBderived vectors containing an N-terminal Myc tag alone or the Myc tag fused to the ZipB coiled-coil segment.…”

“…We have described a general strategy, termed the functional interaction trap (FIT), for forcing the pairwise interaction of two proteins in vivo as a means to elucidate the functional consequences of that interaction (17). We reasoned that this approach could be adapted to induce specific substrate phosphorylation in vivo.…”

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

“…We engineered the HA-frankenbody from six complementarity determining regions (CDRs, or loops) within the heavy and light chains of a published anti-HA scFv (parental full-length antibody: 12CA5) 33,34 (Fig. 1A).…”