Reverse Yeast Two-Hybrid (rY2H) Service for PPI Inhibitor Discovery

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For over two decades, Creative Biolabs has been at the forefront of protein interaction analysis. Our Reverse Yeast Two-Hybrid (rY2H) Service is a powerful, validated platform engineered to identify specific molecules—small molecules, peptides, or biologics—that can effectively dissociate a target protein complex. By leveraging a sophisticated counter-selection strategy, our rY2H system directly links the disruption of a specific PPI to cell survival, enabling robust, large-scale screening campaigns. Partner with us to accelerate your research from target validation to lead identification with unparalleled precision and efficiency.

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What is the Reverse Yeast Two-Hybrid (rY2H) System?

The Reverse Yeast Two-Hybrid (rY2H) system is a powerful genetic method ingeniously adapted from the conventional Yeast Two-Hybrid (Y2H) assay. While Y2H is designed to identify interacting protein partners, the rY2H system is specifically engineered to screen for molecules or mutations that disrupt a known protein-protein interaction.

The core principle relies on a dual-reporter system. First, a positive reporter (e.g., HIS3) confirms the baseline interaction between your "bait" and "prey" proteins, allowing yeast to grow on selective media. Critically, the interaction also activates a counter-selectable reporter gene (e.g., URA3 or CYH2). The expression of this gene product is toxic to the yeast under specific conditions (e.g., in the presence of 5-FOA for URA3).

How It Works

Interaction ON (No Inhibitor): The bait and prey proteins interact, forming a functional transcription factor. This complex activates the toxic reporter gene, leading to cell death on the counter-selective medium.
Interaction OFF (Inhibitor Present): A small molecule or peptide from the screening library successfully binds to one of the partner proteins and disrupts their interaction. The transcription factor cannot form, the toxic reporter gene is not expressed, and the yeast cell survives and proliferates.

This elegant survival-based selection makes rY2H an ideal platform for high-throughput screening (HTS) of vast compound libraries to find your next lead candidate.

Fig. 1 Reverse yeast two-hybrid (rY2H) for drug screening.¹

Our Streamlined rY2H Service Workflow

We follow a meticulous, milestone-driven process to ensure the success of your project.

Step 1: Project Consultation & Design

In-depth discussion with our experts to understand your target PPI and project goals.
Strategic planning for bait and prey construct design, vector selection, and library choice.

Step 2: Bait & Prey Plasmid Construction

Gene synthesis and subcloning of your target proteins into our optimized rY2H vectors.
Sequence verification and quality control.

Step 3: Assay Feasibility & Optimization

Transformation into our proprietary yeast strain.
Confirmation of bait-prey interaction using positive reporter genes.
Validation of the counter-selection system and optimization of screening conditions (e.g., 5-FOA concentration).

Step 4: High-Throughput Screening (HTS)

Large-scale library screening using our automated platform.
Incubation on dual-selective media to isolate survivor colonies (primary hits).

Step 5: Hit Identification & Confirmation

Isolation of primary hits.
Re-streaking and re-testing to confirm the interaction-disrupting phenotype.
"Bait-dependency" and "prey-dependency" tests to eliminate false positives.

Step 6: (Optional) Downstream Validation & Analysis

Dose-response curve generation to determine IC50 values.
In vitro validation using orthogonal methods (e.g., ELISA, SPR, FRET).
Compound identification (if using plasmid-encoded peptide libraries).

Step 7: Comprehensive Data & Reporting

A detailed final report including all methods, raw data, data analysis, hit lists, and expert interpretation.

Applications of the rY2H Service

Our rY2H platform is versatile and can be applied across various stages of the therapeutic development pipeline.

High-Throughput Small Molecule Screening: Screen our extensive, diversity-oriented small molecule libraries (including FDA-approved drugs, natural products, and kinase inhibitors) to identify novel inhibitors for your PPI target of interest.
Therapeutic Peptide and Biologic Discovery: Identify peptides, single domain antibodies, or other biologics that specifically disrupt a target interaction. This is ideal for developing highly specific and potent next-generation therapeutics.
Target Validation and Drug Repurposing: Rapidly validate whether a specific PPI is a viable drug target by demonstrating its druggability. Screen approved drug libraries to find new indications for existing compounds.
Mapping Protein Interaction Interfaces (Alanine Scanning): Utilize rY2H with site-directed mutagenesis to pinpoint critical amino acid residues (hotspots) at the binding interface that are essential for the interaction.
Off-Target Effect and Selectivity Profiling: Assess whether a lead compound disrupts homologous or related PPIs, providing crucial data on its selectivity profile early in the discovery process.

Advantages of Creative Biolabs' rY2H Platform

Choosing Creative Biolabs gives you access to a state-of-the-art platform refined by decades of expertise.

Unmatched High-Throughput Capability: Our fully automated platform, incorporating robotic liquid handling and colony picking, allows for the screening of millions of compounds with exceptional speed and reproducibility.
Customizable and Diverse Libraries: We offer a comprehensive collection of screening libraries and can also screen your proprietary in-house libraries. Our expertise extends to small molecules, linear peptides, and fragment libraries.
Optimized Yeast Strains for High Sensitivity: We utilize proprietary yeast strains engineered for low background noise and high sensitivity, ensuring the detection of even weak inhibitors and minimizing false negatives.
Rigorous Multi-Step Validation: Hits identified from the primary rY2H screen are not the end of the story. We perform a cascade of validation and confirmation steps, including re-testing, counter-screens for cytotoxicity, and dose-response analysis to eliminate false positives and artifacts.
End-to-End Project Management: From initial consultation and construct design to final data analysis and hit validation, a dedicated Ph.D.-level scientist will guide your project, providing regular updates and expert insights.

Explore Our Related Protein Interaction Services

Leverage our full suite of protein interaction and drug discovery services to advance your research. Explore our full range of PPI services here: Protein-Protein Interaction (PPI) Assay Services

Connect with our team of Ph.D.-level experts to discuss your project. We will provide a customized strategy tailored to your specific research goals.

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Frequently Asked Questions (FAQ)

Q: What is the main difference between Y2H and rY2H?

A: Y2H is a discovery tool used to find unknown protein partners ("what does my protein bind to?"). rY2H is a screening tool used to find molecules that disrupt a known protein interaction ("what compound can break this specific protein pair apart?").

Q: What types of protein interactions are suitable for the rY2H system?

A: The rY2H system is ideal for studying binary interactions that can be reconstituted in the yeast nucleus. It is compatible with proteins from various organisms and subcellular locations, though some modifications may be required for membrane or secreted proteins.

Q: How do you handle false positives in the rY2H screen?

A: We employ a rigorous, multi-tiered validation strategy. This includes re-testing hits, performing counter-screens to identify compounds that are generally toxic to yeast or inhibit transcription/translation, and running dependency assays to ensure the effect is specific to the bait-prey interaction.

Q: Can I screen my own compound library?

A: Absolutely. We are happy to work with our clients to screen their proprietary in-house libraries. Please contact us to discuss the logistics and formatting requirements.

Reference

Suter, Bernhard, Saranya Kittanakom, and Igor Stagljar. "Interactive proteomics: what lies ahead?." Biotechniques 44.5 (2008): 681-691. Distributed under Open Access license CC BY 4.0. The image was modified by extracting and using only part of the original image. https://doi.org/10.2144/000112799

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.