Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Abstract

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed "yeast surface two-hybrid (YS2H)," to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two ␣-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pM to 10 M. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pM to 100 M. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chainonly antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA) 2 (2-6),where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7,8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction "hub" according to their binding affinity. Quantitative estimation of protein-protein interactions in vivo will req...

High-throughput engineering and analysis of peptide binding to class II MHC

“…Adaptation of this approach for screening peptide libraries to identify T cell stimulatory ligands (28,35,36) should be straightforward. Furthermore, whereas we have focused on characterizing and engineering MHC-II/peptide recognition, the approach is amenable to extension to a host of other systems for quantitative characterization of protein-protein and proteinpeptide-binding specificities, complementing other methods for studying such interactions (37,38) and broadening the impact of the method.…”

Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies

“…In YS2H, the prey proteins exist on cell surface due to their binding to the bait as well as in culture media; the level of soluble protein in the media was estimated to be around 10 nM for scFv (Fig S3) (20). Antibody concentration in the media was high enough for flow cytometry application without the need for purification.…”

“…Single chain antibodies AM01 and AM17 both (21) is subjected to directed evolution to isolate mutations to induce active conformation, to which a phage library of human single-chain antibody is screened. Final selection of specific antibodies is made in yeast surface 2-hybrid system (20) according to quantitative assessment of monomeric antigen-antibody interactions.…”

“…Then phage library were panned against the active I domain in yeast, where the enrichment of reactive phage clones was monitored by flow cytometry. From enriched phage libraries, cDNAs encoding single chain fragment variable (scFv) antibody and active Mac-1 I domain were introduced as a pair of the bait and prey proteins into the yeast surface two-hybrid system (YS2H) (20) that was designed for quantitative estimation of protein-protein interactions. Antibodies selected from two steps of phage binding to yeast and antigen-antibody interactions in YS2H were found to react with the active Mac-1 I domain and be potent in blocking neutrophil adhesion and migration.…”

Engineering of Single Ig Superfamily Domain of Intercellular Adhesion Molecule 1 (ICAM-1) for Native Fold and Function

“…ICAM-1 of varying lengths, residues Gln-1 to Trp-84 (D1), residues Gln-1 to Leu-187 (D1D2), and residues Gln-1 to Arg-451 (D1-D5) were subcloned into the display plasmid named CAga2, which was modified from pCTCON (35) to express Aga2 at the C-terminal of the protein of interest (36 Error-prone and Focused Mutagenesis Libraries-ICAM-1 cDNA library was constructed from error-prone PCR amplification of ICAM-1 cDNA using GeneMorph II Random Mutagenesis kit (Stratagene). In focused mutagenesis, four primers spanning ICAM-1 cDNA were synthesized with mixed nucleotides into selected positions for the substitution of a subset of 20 amino acids (described in detail under "Results").…”