Plant Yeast Two-Hybrid Library Construction and Screening Service
Plant Protein-Protein Interaction Screening
CD BioSciences provides Plant Yeast Two-Hybrid (Y2H) library construction and screening services for identifying candidate proteins that interact with a plant bait protein.
The service can support bait assessment, bait vector construction, self-activation and toxicity testing, prey cDNA library construction or selection, library screening, positive clone confirmation, sequencing, and hit analysis. Nuclear Y2H and membrane-oriented Y2H strategies can be considered according to the bait protein type.
Principle of Y2H
In a conventional nuclear Y2H system, the bait protein is fused to a DNA-binding domain and prey proteins are fused to a transcriptional activation domain. If bait and prey interact in yeast, the two domains are brought together and reporter gene expression is activated under selective conditions.
Y2H screening is a discovery method for candidate protein-protein interactions. Positive hits should be confirmed by re-transformation or point-to-point validation and, when needed, by orthogonal assays such as Co-IP, GST pull-down, BiFC, LCA, or in planta functional tests.
Figure 1. Y2H principle showing bait-prey interaction and reporter activation.
Service Scope
Protein sequence, localization, transmembrane region, signal peptide, toxicity, and self-activation risk can be evaluated before screening.
Plant tissue or cell samples can be used to construct prey cDNA libraries for Y2H screening.
Bait constructs are screened against prey libraries under optimized selection conditions.
Positive clones can be confirmed by colony PCR, sequencing, annotation, and point-to-point Y2H validation.
Nuclear Y2H and Membrane Y2H
Conventional nuclear Y2H is suitable for soluble proteins that can function in a yeast nuclear reporter context. Membrane proteins or proteins with strong transmembrane regions may require a membrane yeast two-hybrid strategy based on split-ubiquitin logic. Choosing the correct system helps reduce false negatives caused by inappropriate bait localization or topology.
Figure 2. System selection for nuclear Y2H and membrane Y2H projects.
Workflow
Figure 3. General workflow for plant Y2H library construction and screening.
- Review of bait protein sequence and project objective
- Bait vector construction and yeast transformation
- Self-activation and toxicity testing
- Prey cDNA library construction or library selection
- Library screening under selective conditions
- Positive colony picking and PCR confirmation
- Sanger sequencing, hit annotation, and report delivery
Controls and Limitations
| Item | Why It Matters |
|---|---|
| Bait self-activation test | Prevents reporter activation by bait alone from being misread as interaction. |
| Bait toxicity test | Detects bait constructs that impair yeast growth and reduce screening efficiency. |
| Prey library QC | Library complexity, insert size, and positive rate affect screening coverage. |
| Point-to-point validation | Re-confirms candidate bait-prey pairs outside the initial library screen. |
Y2H detects interactions in a yeast reporter environment. It may miss interactions requiring plant-specific modifications, cofactors, localization, or complex assembly conditions.
Sample and Information Requirements
- Bait gene or protein sequence
- Species and tissue/cell source for prey library construction
- Known domains, transmembrane regions, or localization information
- Existing bait plasmid, if available
- Number of bait proteins and screening depth requirements
- Expected follow-up validation design
Deliverables
- Bait vector construction information
- Self-activation and toxicity test records
- Library QC information, if library construction is included
- Screening images and experimental records
- Positive clone PCR and sequencing results
- Hit annotation table and standard project report
Related Services
Please contact us with your bait protein sequence, plant sample source, and screening objective for project evaluation.
For research use only, not for clinical use.