Isolation of receptor–ligand pairs by capture of long-lived multivalent interaction complexes

Abstract

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab-antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab-antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor-ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.I nteractions among extracellular proteins, such as receptorligand interactions, play a crucial role in the development, cell-to-cell communication, and immune defense of multicellular organisms. Yet, standard technologies for the analysis of protein-protein interactions such as the two-hybrid system (1) but also more recent strategies such as the protein-complementation assay approaches (2-5) and RNA polymerase recruitment (6) operate inside the cell. Although there are examples of successful applications of these systems to extracellular proteins, frequently such proteins (especially those with disulfide bridges), do not attain full functionality when expressed in the reducing environment of the cytoplasm.Because of the importance of extracellular protein-protein interactions for understanding biological function as well as for drug discovery, many useful methods have been developed for their analysis and selection. The most widely used are the various protein-display technologies (reviewed in ref. 7). Phage display in particular has allowed the isolation of many novel peptide and protein ligands as well as the isolation of Abs directly from diverse libraries of V genes (7). Current display technologies are mostly used for the isolation of ligands to a single target, available in at least partially purified form and linked to a solid support. To increase throughput and to increase the utility of display methods to proteomics applications, it would be desirable to be able to select directly for interacting receptor-ligand pairs starting from their encoding genes or repertoires thereof. Within phage display several approaches (called SAP, SIP, and CLAP; reviewed in ref. 8) have been described, which rely on reconstitution of phage infectivity by cognate interactions and allow selection of protein-protein interaction pairs. However, reconstitu...

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“…Another consequence of the burgeoning genomic data is the absence of suitable analytical reagents such as antibodies to investigate the predicted proteome, due to the low‐throughput of conventional antibody development protocols. Many groups have developed novel technologies to address this situation, involving combinatorial yeast‐phage display, selectively infective phage, protein fragment complementation assays (PCAs) and selection by avidity capture (SAC) 10–13. These techniques rely on simultaneous combinatorial expression of antigen and antibody libraries and conditional enzymatic activity or fluorescence‐activated cell sorting (FACS) for selection of cognate pairs.…”

“…Joining interacting genes to form a single segment of DNA naturally preserves interaction information. However, this method requires modification of existing yeast plasmid libraries and strains and is not applicable to other display systems such as yeast‐phage display 10 or selection by avidity capture 13. Furthermore, it would be desirable to preserve cognate pairing information in naturally expressed gene pairs such as antibody heavy and light chains 15.…”

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

“…The AVEXIS method described here provides a sensitive method for detecting transient extracellular protein:protein interactions that can be implemented in a scalable manner with a low false positive rate. While other scalable methods have been developed to detect extracellular interactions [11][12][13][14][15] , one advantage of AVEXIS is that the experimental parameters such as the bait and prey activities have been quantified to detect even the weakest of interactions (t 1/2 ≤ 0.1 s) while retaining a low false positive rate 3 . In addition, the streamlined and robust sample preparation procedures have enabled this assay to be implemented on a much larger scale than other assays and we have recently described a systematic interaction screen totalling over ~16,500 potential interactions 16 .…”

Multivalent scFv Display of Phagemid Repertoires for the Selection of Carbohydrate-specific Antibodies and its Application to the Thomsen–Friedenreich Antigen

“…30 No significant differences in the display level could be detected, and all constructs seemed to be associated with similar amounts of non-fused scFv (Supplementary Material, Figure 2). …”