Yeast display and phage display are two powerful technologies used for protein engineering, antibody discovery, and molecular interaction studies. Both involve displaying proteins, peptides, or antibody fragments on the surface of a biological system (yeast or phage) and screening for high-affinity binders, but they differ in several key aspects. Here’s a comparison of yeast display and phage display:
Biological System
Yeast Display
Uses _Saccharomyces cerevisiae_ (often strain EBY100) or other yeast species.
Proteins or peptides are displayed on the yeast cell surface, typically by fusing them to a cell wall anchor protein, such as Aga2p, which binds to the Aga1p protein on the yeast cell wall.
Yeast cells are eukaryotic, allowing for post-translational modifications (e.g., glycosylation, proper folding) of the displayed proteins.
Phage Display
Uses bacteriophages, usually M13 filamentous phage, as the display system.
Proteins or peptides are fused to one of the phage coat proteins (e.g., pIII or pVIII), which display the proteins on the surface of the phage particle.
Phage display is based on prokaryotic systems (bacteria), meaning that proteins are expressed in Escherichia coli (E. coli) before being displayed on the phage surface. This system does not provide eukaryotic post-translational modifications.
Post-Translational Modifications
Yeast Display
Yeast, being eukaryotic, can perform post-translational modifications like glycosylation, disulfide bond formation, and proper protein folding, which is important for the function and stability of complex eukaryotic proteins (e.g., antibodies, receptors).
Glycosylation in yeast differs from mammalian cells, which can sometimes be a disadvantage if human-like glycosylation is essential for the activity of the displayed protein.
Phage Display
As a bacterial system, phages do not offer eukaryotic post-translational modifications. Proteins that require glycosylation, disulfide bonds, or complex folding may not fold correctly when displayed on phage surfaces.
This limits the ability to display some mammalian proteins and complex molecules.
Screening and Selection
Yeast Display
Selection is typically performed using fluorescence-activated cell sorting (FACS). This allows for quantitative, high-resolution sorting based on the fluorescence intensity of labeled targets, enabling precise selection of yeast cells displaying proteins with high affinity for the target.
Yeast display can be used for iterative rounds of sorting to enrich high-affinity binders, allowing researchers to select for both affinity and expression levels.
Phage Display
Selection is performed using biopanning, where phage particles displaying proteins are exposed to a target (e.g., immobilized on a plate), and those that bind to the target are retained while non-binders are washed away.
Multiple rounds of biopanning help enrich for phages displaying proteins with high affinity for the target, but this method is often less quantitative compared to yeast display with FACS.
Library Size
Yeast Display
Yeast can accommodate libraries of up to 10⁶ to 10⁹ variants, depending on the transformation efficiency. While this is lower than phage display, yeast display still allows for the screening of diverse libraries.
The lower library size is compensated by the high screening precision provided by FACS.
Phage Display
Phage display can handle very large libraries, typically ranging from 10⁹ to 10¹¹ variants or even larger.
The high library size allows for the screening of massive diversity, which is particularly useful for early-stage discovery or when screening highly diverse peptide or antibody fragment libraries.
Ease of Use and Throughput
Yeast Display
Requires specialized equipment like FACS for high-throughput sorting, which provides highly quantitative data but requires access to a flow cytometer.
Yeast culture conditions are relatively straightforward, but expression levels may need optimization, especially for large or complex proteins.
Phage Display
Easier to set up initially as it relies on biopanning, which doesn’t require specialized equipment (just immobilized targets and washing steps).
Higher throughput in terms of the number of variants that can be screened quickly, but lacks the high-resolution screening capability of FACS.
Types of Proteins Displayed
Yeast Display
Works well for displaying large, complex proteins (e.g., full-length antibodies, receptors, enzymes) due to the eukaryotic system’s ability to fold and modify these proteins correctly.
It is particularly useful for antibody discovery, affinity maturation, and screening of protein-protein or protein-peptide interactions.
Phage Display
Commonly used for displaying small peptides, antibody fragments (e.g., single-chain variable fragments, scFv; Fab fragments), or relatively simple proteins.
While phage display can handle small peptides well, complex proteins or full-length antibodies may not fold properly.
Protein Size and Complexity
Yeast Display
Can handle large and complex proteins, including full-length antibodies, extracellular domains of membrane proteins, or multi-domain enzymes, because of its eukaryotic folding and modification capabilities.
Phage Display
Works well for small peptides and simpler proteins, but larger proteins or those requiring post-translational modifications may not fold properly and therefore may not function optimally when displayed on the phage surface.
Applications
Yeast Display
Antibody discovery and engineering: Yeast display is particularly useful for affinity maturation, epitope mapping, and antibody engineering because it allows for high-precision sorting.
Protein engineering: Yeast display is effective in screening libraries of proteins for improved binding, stability, or enzymatic activity.
Receptor-ligand interactions: The ability to display large, complex proteins makes yeast display suitable for studying receptor-ligand interactions
Phage Display
Antibody discovery: Phage display is widely used for the initial discovery of antibody fragments (e.g., scFvs, Fabs) from large libraries due to the system’s ability to handle extremely large libraries.
Peptide display: Phage display is ideal for discovering peptide binders or mimotopes that mimic epitopes recognized by antibodies.
Protein-protein interactions: Phage display is also used for discovering protein-protein interaction domains, especially when screening small, stable protein fragments.
Affinity and Selection Precision
Yeast Display
Provides high precision in affinity selection due to FACS, which can distinguish small differences in binding affinity, making it an excellent choice for fine-tuning protein interactions.
Allows for quantitative analysis of protein binding affinities in real time during the selection process.
Phage Display
Offers good initial screening for binding but lacks the precision of yeast display in distinguishing small affinity differences.
Typically used for broad selection of binders, which may require further optimization in later stages of development.
Speed and Iteration
Yeast Display
Can be slower to develop due to the complexity of yeast culture, expression induction, and FACS-based sorting, especially when working with larger proteins.
However, real-time affinity measurement during FACS sorting can save time in downstream validation.
Phage Display
Faster for initial rounds of selection and screening due to the simple biopanning process, which can be completed quickly.
Iterative biopanning can take several rounds to yield high-affinity binders, followed by downstream validation steps.
Conclusion: Yeast Display vs. Phage Display
Yeast display is ideal for applications that require high precision and screening of complex or large proteins, making it especially useful for antibody engineering, affinity maturation, and eukaryotic protein interactions. Its strength lies in the ability to perform quantitative screening and handle proteins requiring post-translational modifications.
Phage display excels in high-throughput screening of large libraries, particularly for smaller, less complex proteins or peptides, and is widely used in early-stage antibody discovery and peptide screening. It’s faster for initial screening but may require additional rounds of optimization and validation for complex targets.
The choice between yeast display and phage display depends on the specific application, the size and complexity of the protein being studied, the need for post-translational modifications, and the level of precision required in the selection process.