Yeast display sorting is a critical step in the yeast surface display process, used to identify and isolate yeast cells displaying peptides or proteins with desirable binding properties, such as high affinity or specificity for a target molecule. This process relies heavily on fluorescence-activated cell sorting (FACS), which allows researchers to quantitatively measure the binding of displayed proteins or peptides to fluorescently labeled targets and then isolate the cells that exhibit the strongest binding.
Overview of Yeast Display Sorting Process
Expression of Protein/Peptide Libraries on Yeast Surface
The yeast cells display a library of protein or peptide variants on their surface, typically fused to a surface anchor protein like Aga2p. These variants could be antibodies, enzymes, peptides, or other protein fragments.
Binding to Target Molecule
Yeast cells are incubated with a fluorescently labeled target (e.g., a protein, receptor, antigen, or small molecule). Yeast cells displaying peptides or proteins that bind to the target will retain the fluorescent label on their surface.
Fluorescence-Activated Cell Sorting (FACS)
After incubation with the labeled target, the yeast cells are passed through a flow cytometer, where cells are sorted based on their level of fluorescence. Cells displaying proteins or peptides that bind strongly to the target exhibit higher fluorescence intensity and are separated from those with weaker or no binding.
Selection and Enrichment of High-Affinity Binders
The yeast cells with the highest fluorescence intensity (i.e., those displaying the best binders) are collected and cultured. This population can then undergo multiple rounds of sorting to further enrich for variants with even higher affinity or specificity.
Steps in the Yeast Display Sorting Process
Preparation of Yeast Cells Displaying Protein/Peptide
Induce Surface Display: Yeast cells containing the library are cultured under conditions that induce the expression of the gene encoding the protein or peptide of interest fused to the Aga2p surface anchor. This results in the display of these proteins/peptides on the surface of the yeast cells.
Incubation with Labeled Target
The yeast cells are incubated with a fluorescently labeled target. The target can be labeled with a fluorescent dye such as FITC, PE, or Alexa Fluor. If the protein or peptide displayed on the yeast surface binds to the target, the yeast cells will be fluorescently labeled.
Secondary Labeling (Optional): In some cases, a secondary fluorescent antibody may be used to detect the protein of interest or specific epitope tags like FLAG or HA, to confirm surface display of the fusion protein.
Fluorescence-Activated Cell Sorting (FACS)
Sorting Based on Fluorescence:
The yeast cells are passed through a flow cytometer, where each cell’s fluorescence is measured as it flows through a laser beam. The cells are sorted into different populations based on the intensity of the fluorescence signal.
Dual-Color Sorting: Often, two fluorescent signals are measured:
Protein Display: One fluorescent signal is used to confirm that the protein or peptide is being properly displayed on the yeast surface (e.g., through an epitope tag like HA or FLAG).
Target Binding: The other fluorescent signal is used to measure the binding of the displayed protein or peptide to the fluorescently labeled target.
Gating for Selection
Researchers set a gate to select yeast cells that show both high levels of surface protein display and strong binding to the target. These cells are separated from the rest of the population and collected for further rounds of sorting or analysis.
Negative Sorting (Optional): If specificity is a concern, yeast cells can also be incubated with an irrelevant target or labeled non-target molecule to eliminate non-specific binders. Cells that bind to the non-target molecule are excluded from the final selection.
Enrichment of High-Affinity Binders
Multiple Rounds of Sorting:
The yeast cells collected after the first round of sorting can be expanded in culture and subjected to additional rounds of sorting. Each round refines the population, enriching for yeast cells displaying peptides or proteins with higher affinity or specificity for the target.
Typically, 2–4 rounds of sorting are performed, with progressively narrower gates in FACS to select the highest affinity binders.
Analysis and Recovery of Selected Clones
Sequencing of Selected Variants:
After sorting, the DNA encoding the protein or peptide from the selected yeast clones is recovered and sequenced to identify the sequences of the high-affinity binders.
Next-Generation Sequencing (NGS): NGS can be used to analyze the entire population of binders, allowing for detailed insights into the diversity of selected variants and the frequency of specific mutations.
Functional Validation:
The selected clones can be subjected to further biochemical and functional assays to validate the binding affinity and specificity. Common methods include surface plasmon resonance (SPR), enzyme-linked immunosorbent assays (ELISA), and competition binding assays.
Applications of Yeast Display Sorting
Antibody Engineering and Selection
Yeast display sorting is widely used in antibody discovery and engineering to identify antibody fragments (such as scFvs or Fabs) with high affinity for antigens. By sorting libraries of antibody variants, researchers can isolate clones that bind tightly to a target antigen.
Protein-Protein Interaction Studies
Yeast display sorting is used to study protein-protein interactions by screening peptide or protein libraries for variants that bind specifically to a target protein.
Directed Evolution and Protein Engineering
Through iterative rounds of yeast display sorting, proteins can be evolved to improve properties like binding affinity, stability, or enzymatic activity. This method is frequently applied to optimize therapeutic proteins, enzymes, and receptors.
Peptide Display and Screening
Peptide libraries displayed on yeast can be sorted to identify binding peptides that interact with a target protein, receptor, or other molecules. These peptides can be used in therapeutic development or as molecular tools to study biological pathways.
Epitope Mapping
Yeast display sorting can be used to identify the specific regions (epitopes) of a protein that are recognized by antibodies. By displaying fragments or peptides of the target protein on yeast and sorting based on antibody binding, epitope mapping can be performed.
Drug Discovery
Yeast display libraries can be used to screen for small peptides or protein domains that bind to drug targets, providing potential leads for therapeutic development.
Advantages of Yeast Display Sorting
Quantitative Analysis: FACS allows for precise, quantitative measurement of binding interactions, enabling the selection of the best binders with the highest affinity.
High-Throughput Screening: Yeast display sorting enables the screening of large libraries of protein or peptide variants in a high-throughput manner, making it ideal for directed evolution and protein engineering.
Eukaryotic System: Yeast cells provide a eukaryotic environment for protein folding and post-translational modifications, making yeast display more biologically relevant for certain proteins than bacterial display systems.
Multiple Rounds of Enrichment: The ability to perform multiple rounds of sorting allows for the progressive enrichment of clones with higher affinity or specificity, leading to the selection of the best possible variants.
Challenges and Limitations of Yeast Display Sorting
Glycosylation Differences: Yeast glycosylation patterns differ from those of mammalian cells, which may affect the binding or activity of certain glycosylated proteins.
Size Limitations: Larger proteins or complex protein structures may not display efficiently on the yeast surface, which can limit the types of proteins that can be screened using this method.
Off-Target Binding: Non-specific binding can sometimes occur, especially when the target protein is present in high concentrations. Negative sorting steps can mitigate this, but careful optimization is needed.
Conclusion
Yeast display sorting, particularly using FACS, is a powerful tool for identifying high-affinity protein and peptide binders from large libraries. By enabling high-throughput and quantitative screening, this method has revolutionized protein engineering, antibody discovery, and functional genomics. Through multiple rounds of sorting, yeast display allows for the enrichment of clones with optimal binding properties, making it a critical method for directed evolution and drug discovery.