Yeast surface display technology is a versatile platform applied in fields such as antibody discovery, target identification, and protein engineering.
In antibody engineering, this technology leverages the characteristics of the yeast eukaryotic expression system to display antibody library proteins on the yeast cell surface. Screening is then performed using magnetic beads and flow cytometry to isolate antibody sequences with high affinity or stability. The technology is widely applied to various antibody formats—including scFv, Fab fragments, full-length IgG, and camelid VHH (single-domain antibodies)—as well as to processes such as affinity maturation, stability enhancement, and pH stability optimization. AlpVHHs possesses a mature and stable yeast display platform, enabling the rapid and efficient screening and optimization of peptides and antibodies.
While various yeast strains and cell wall anchoring sites have been developed to display a wide range of proteins, the most classic and widely used system is the Aga1p-Aga2p system based on *Saccharomyces cerevisiae*: [Yeast Cell Wall] — (Covalent) —> Aga1p === (Disulfide Bond) ===> Aga2p ——> [Target Protein (POI)]
![[Yeast Surface Display Series①] Getting Started from Scratch: An Analysis of Core Principles and Technical Advantages](data/watermark/main/ueditor/20260806/6a743122b1ea0.png "[Yeast Surface Display Series①] Getting Started from Scratch: An Analysis of Core Principles and Technical Advantages")
● Anchoring Core: The agglutinin Aga1p, secreted by yeast cells, is firmly anchored to the cell wall via covalent bonds (linked to β-glucan).
● Bridging Link: The gene encoding the target protein is cloned into an expression vector and fused to either the C-terminus or N-terminus of Aga2p.
● Protein Display: Aga2p associates with Aga1p intracellularly via two disulfide bonds, ultimately "pushing" the target protein to the cell's outer surface through the secretory pathway.
● Display Verification: Distinct epitope tags (such as HA and c-myc) are typically introduced at both ends of the target protein. Fluorescently labeled antibodies are then used to separately quantify "display efficiency" and "target binding capability."
Compared to phage display, yeast display offers the following significant advantages:
1. Yeast possesses a secretory pathway similar to that of higher eukaryotes; protein folding occurs in the endoplasmic reticulum, where chaperones, folding enzymes, and quality-control mechanisms ensure that only correctly folded proteins are secreted. In contrast, phage display relies on a prokaryotic system; certain antibody clones can be toxic to *E. coli*, leading to slow growth or even a lack of growth, which results in library bias and the loss of clones.
2. Screening for high-affinity antibodies via phage display is often adversely affected by the screening process itself, as the outcome depends on antibody expression levels in addition to affinity. Yeast display systems utilize FACS (Fluorescence-Activated Cell Sorting) to screen based on both antibody affinity and display levels; this eliminates expression-related bias and enables the discrimination of clones with very subtle differences in affinity.
3. Yeast display is a multivalent display system, allowing for the simultaneous sorting of high-, medium-, and low-affinity clones in a single run, offering great convenience.
4. Dual-staining FACS allows for the direct determination of antibody affinity on the yeast cell surface, eliminating the need for time-consuming subcloning, expression, and purification steps.
5. Antibody screening is rapid, sorting 100,000 cells takes just three minutes and yields over 100 unique candidate clones.
6. Antibody affinity and stability can be optimized within the same display system.
● Fragment Preparation: Antibody gene fragments (e.g., scFv, VHH, or Fab) are amplified via PCR, incorporating homology arms (typically 40–50 bp) at both ends that match the yeast expression vector.
● Homologous Recombination: *Saccharomyces cerevisiae* possesses robust *in vivo* homologous recombination capabilities. By co-electroporating the linearized vector backbone and the amplified antibody fragments into competent yeast cells (e.g., EBY100), the yeast cells assemble the complete expression plasmid *in vivo*. This greatly simplifies the *in vitro* ligation process, enabling library sizes to easily reach 10^7–10^9.
● Transcriptional Regulation: The system utilizes a galactose-inducible promoter. After cells are expanded to the logarithmic growth phase in glucose-containing medium (SD-CAA), the culture is switched to galactose-containing medium (SG-CAA) to induce expression of the target protein.
● Flow Cytometric Analysis: The antibody is fused with a downstream expression tag, allowing for the quantitative monitoring of total antibody expression levels using fluorescently labeled antibodies.
● Primary Screening (MACS): Magnetic-Activated Cell Sorting (MACS) is typically employed for the initial round. Biotinylated antigens are conjugated to magnetic beads to rapidly filter out approximately 99% of non-specific clones.
● Secondary Screening (FACS): Fluorescence-Activated Cell Sorting (FACS) is subsequently used. Dual-color fluorescence staining is applied: Channel A measures the epitope tag (indicating display level), while Channel B measures antigen binding (indicating affinity).
The yeast surface display platform offers distinct advantages for antibody drug discovery, particularly due to the ability to precisely control the screening process using flow cytometry.
1) Equilibrium Sorting: Screening for high-affinity clones using low antigen concentrations.
2) Off-rate Sorting: Following antigen binding, an excess of unlabeled antigen is added to induce competitive dissociation; variants with extremely slow dissociation rates ($k_{off}$)—indicating highly stable binding—are retained, enabling precise molecular-level evolution.
3) Blocking: Utilizing receptor/ligand competition to screen for antibodies that block receptor-ligand interactions.
4) Epitope Competition Screening: Screening for antibodies that target the same epitope as a reference antibody by using a blocking antigen.
AlpVHHs was the first company in China to establish a yeast display platform. It has provided services to over 300 domestic enterprises for projects including peptide discovery, antibody (VHH, scFv) discovery, affinity maturation, and pH-sensitive antibody engineering based on yeast display technology.
1. Library size > 2 × 10⁸; library expression efficiency > 60%
2. Over 500 projects completed; a mature and stable platform
