Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Executive Industry Relevance

Interactome-Seq enables unbiased, high-throughput identification of functional protein domains from any DNA source, supporting target validation and mechanistic de-risking in early discovery. By integrating phage display with next-generation sequencing, the method provides quantitative, reproducible data on domain abundance and interactions, enhancing predictive confidence in lead identification. This approach addresses the need for scalable, reproducible systems to prioritize targets and reduce biological risk in protein-based therapeutic development.

Strategic Applications in Biopharma R&D

Early Discovery & Target Validation

• Scientific Value: Enables interrogation of therapeutic hypotheses by isolating correctly folded, soluble protein domains from genomic DNA for functional annotation.
• Operational Value: Provides an unbiased, high-throughput method to assess domain functionality across entire genomes without prior bias.
• Predictive Value: Supports predictive confidence by identifying domains with desired binding properties to antibodies or proteins, informing target selection.

Screening & Assay Development

• Scientific Value: Generates validated domainome libraries suitable for phage display screening against therapeutic targets such as antibodies or binding proteins.
• Operational Value: Enables standardized library preparation with quantifiable outputs via NGS, supporting assay reproducibility and scalability.
• Screening Readiness: Facilitates preparation of diverse, soluble domain fragments for downstream screening campaigns with defined diversity and abundance metrics.

Translational & Preclinical Research

• Translational Continuity: Supports mechanistic de-risking by linking domain identification to structural and functional studies, enabling progression from discovery to preclinical validation.
• Biomarker Alignment: Enables antigen identification and interaction mapping relevant to antibody target validation and immuno-oncology applications.
• Risk-Adjusted Advancement: Provides quantitative NGS data on enriched clones to inform go/no-go decisions based on domain abundance and interaction profiles.

Pipeline & Workflow Integration

The method integrates into the discovery continuum from early target identification through lead optimization, providing domain-level resolution for protein interaction networks and antigen profiling.

• Discovery Biology: Supports hypothesis testing and pathway clarification by isolating functional domains from complex DNA sources for interaction mapping.
• Screening: Enables assay-ready library construction with quantifiable diversity and abundance, facilitating reproducible phage display campaigns.
• Analytics: Delivers precise mapping, abundance quantification, and diversity metrics via NGS, enabling data-driven comparison of selected fragments under different conditions.
• Translational Research: Connects domain identification to structural and functional characterization, supporting continuity into preclinical validation of protein interactions and antigen signatures.
• Enterprise Reuse: Establishes a reusable platform for domainome generation applicable across multiple targets, projects, and therapeutic areas without re-engineering.

Operational & Enterprise Impact

• Scientific Value: Increases target validation confidence by reducing mechanistic ambiguity through unbiased domain selection and functional screening.
• Operational Value: Ensures standardization and reproducibility via controlled library construction, phage display selection, and NGS-based quantification.
• Strategic Value: Improves go/no-go decision-making by providing quantitative interaction data, reducing late-stage biological failure risk.
• Portfolio Impact: Enables risk-adjusted prioritization of targets based on domainome-derived interaction profiles and abundance data.

Implementation Considerations

• Requires expertise in molecular cloning, phage display, and next-generation sequencing workflows.
• Dependent on access to sonication equipment, agarose gel systems, electroporation units, and Illumina sequencing platforms.
• Necessitates cross-team standardization between molecular biology, proteomics, and bioinformatics for consistent library preparation and data interpretation.
• Adaptation across model systems requires validation of folding reporter functionality (e.g., TEM-1 β-lactamase) in diverse genomic contexts.
• Practical limitations include dependence on DNA quality and fragment size distribution, which may affect library diversity and require optimization of sonication and size selection parameters.

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