Yeast display technology was first introduced in 1997 by Boder and Wittrup in the United States, utilizing the principles of yeast surface display, they fused exogenous proteins to proteins on the yeast cell surface, thereby enabling their expression on the yeast surface. Subsequently, George P. Smith and his colleagues refined this technology, for instance, in 1998, Smith et al. reported a simpler and more efficient method for constructing yeast surface display protein libraries. With the continuous improvement and refinement of the technique, yeast display technology has now been widely applied in fields such as protein functional analysis, drug screening, antigen identification, and vaccine development.
Yeast Surface Display is a high-throughput protein screening technique based on a eukaryotic expression system. This technique achieves a direct link between phenotype and genotype by fusing exogenous proteins—such as antibodies, nanobodies, or peptides—with yeast cell wall proteins, thereby displaying the target molecules on the surface of the yeast cells.
With advancements in technology, yeast display has emerged as a crucial tool in the fields of antibody and protein engineering, finding widespread application in:
● Antibody/nanobody screening
● Protein engineering optimization
● Drug target validation
● Vaccine development and antigen identification
Exogenous peptides, antibodies, or proteins are expressed as fusion proteins within a yeast surface display system, thereby enabling their efficient display on the yeast cell surface. This technology typically utilizes two yeast strains: Saccharomyces cerevisiae and Pichia pastoris. Saccharomyces cerevisiae is commonly employed for the expression of proteins and antibodies, whereas Pichia pastoris is better suited for the expression of complex glycosylated proteins.
Commonly used anchoring proteins include α-agglutinin, Flo1p, Yps1p, Cwp2p, Sed1p, Pir1–4, and others (Fig. 2). The displayed proteins traverse the secretory pathway: they are first transported into the lumen of the endoplasmic reticulum, then transported from the endoplasmic reticulum to the Golgi apparatus, and are ultimately anchored to the cell wall surface.
Yeast display system inherits the key characteristics of phage display—namely, the direct linkage between phenotype and genotype, and ease of amplification—thereby enabling the screening of target genes based on the specific properties of the encoded proteins. In addition to utilizing traditional biopanning methods for screening, the yeast display system possesses a distinct advantage stemming from the relatively large size of yeast cells: it allows for screening via Fluorescence-Activated Cell Sorting (FACS). The specific procedure is as follows: the display library is incubated with the target molecule; yeast cells expressing ligands for the target molecule on their surface will bind to it, while unbound target molecules are subsequently washed away. Next, fluorescently labeled antibodies are added, and any unbound antibody molecules are removed by washing. Finally, the suspension of fluorescently labeled cells is subjected to FACS to sort and isolate the yeast cells that express the target protein.
Yeast Surface Display Platform by AlpVHHs employs an "α-agglutinin" display system, in which the gene sequences for nanobodies and the HA tag are inserted into a display plasmid vector utilizing the Aga2 protein scaffold. This vector utilizes nutritional markers to maintain selective growth within the yeast cells; subsequently, the addition of galactose to the culture medium induces the yeast to initiate surface display, resulting in the secretion and anchoring of the nanobody fusion proteins onto the yeast cell walls. By integrating magnetic bead and cell sorting technologies for multiple rounds of screening, high-affinity and highly stable clones can be successfully isolated.
Fig 1. Principle of Yeast Surface Display
Yeast possesses a secretory pathway analogous to that of higher eukaryotes; protein folding occurs within the endoplasmic reticulum, where chaperone proteins, folding enzymes, and quality control mechanisms ensure that only correctly folded proteins are secreted.
Yeast Display system employs FACS sorting technology to screen based on antibody affinity and display level; consequently, it eliminates biases arising from expression and enables the differentiation of clones with even minute differences in affinity.
Screening methods based on yeast surface display are compatible with the quantitative and multiparametric analysis capabilities provided by flow cytometry. This allows for the precise control of conditions—such as buffer composition, pH, temperature, and antigen concentration—enabling real-time monitoring of display levels and antigen-binding characteristics, as well as the sorting of clones that bind to the target protein under specific incubation conditions.
By utilizing multi-stained FACS, antibody affinity can be directly determined on the surface of yeast cells, thereby eliminating the need for time-consuming subcloning, expression, and purification.
Antibody screening is rapid, enabling the sorting of 100,000 cells in just 3 minutes; when combined with NGS, this process yields hundreds of candidate clones with unique sequences.
● Yeast Surface Display demonstrates exceptional performance in the screening of nanobodies (VHH):
● High-affinity antibody screening
● Immune checkpoint targets (PD-1, PD-L1, CTLA-4, etc.)
● Development of highly specific antibodies
● Development of TCR mimic antibodies
AlpVHHs integrates yeast surface display, flow cytometry sorting, and NGS to overcome the limitations of conventional library screening, effectively doubling experimental efficiency. In a single experiment, this approach yields hundreds of high-quality clones—a hundredfold improvement over traditional screening methods. We have successfully screened high-affinity antibodies (targeting HSA, etc.), blocking antibodies (targeting PD-L1, PD-1, CTLA-4, LAG3, TIGIT, etc.), specific antibodies (targeting CD16a, FcRL5, etc.), and TCR-mimic antibodies for numerous enterprises.
Following library construction, VHH candidates are screened using dual-selection:
● MACS (magnetic-activated cell sorting) for initial enrichment
● FACS (fluorescence-activated cell sorting) to isolate specific binders
This dual-step process facilitates the identification of binder proteins with optimal binding properties and functional stability, providing a robust platform for generating lead candidates for downstream applications.
Fig 2. Yeast Surface Display screening Workflow
Service | Delivery | Time |
lLibrary Construction | lYeast Display Library lLibrary QC Report | 4 Weeks |
lLibrary Screening | lSpecific Clones & Sequences lProject Report | 4-6 Weeks |
1. High-quality library construction standards, with a yeast library capacity reaching 109-1011.
2. Optimized signal peptides enable a library expression rate of 70%–80%.
3. High-quality yeast display library, with a capacity of up to 10^8 CFUs.
4. Stable platform with extensive experience.
Our VHH R&D experience using the Yeast Surface Display Platform spans a wide range of targets, including, but not limited to, the following:
● BBB Shuttling Platform | ● IGF-1R, TfR1 |
● ADC Targets | ● MSLN, Trop2, DLL3, GPC3, FOLR1, B7H3 |
● Ophthalmology Targets | ● ANG2, FZD4, LRP5, VEGF-A |
● Metabolic Disease Targets | ● ActRIIA, ActRIIB, C5, GDF15, MSTN |
● Immune Modulator | ● B7H7,CTLA-4, LAG-3, PD-1, PD-L1, BAFF-R, TIGIT |
● Autoimmune Disease Targets | ● TSLP, IL-33, IL-13, IL-17A, IL-23p19, IL-25, IL-4, IL-6 |
In yeast display experiments, library quality directly determines the success rate of the screening process. For researchers seeking to rapidly launch their projects, utilizing a ready-made, high-quality Naïve VHH Library represents a more efficient choice. The features of Large Naïve VHH Library roducts provided by AlpVHHs:
● Library capacity reaching up to 109-1011.
● High diversity coverage.
● High insertion rate: The positive insert ratio was more than 99.99%.
● Optimized design to enhance expression success rates.
Code | Description | Application |
CamSDAb-Y | yeast display library for screening |
Our high-diversity large naïve VHH library from Camel for antibody discovery. Yeast display from Camel enables screening of recombinant single-domain antibodies (VHHs) with subnanomolar affinity, suitable for FACS, ELISA, WB, and therapeutic research applications.
In addition to yeast display platform development and high-quality VHH libraries, AlpVHHs also offers comprehensive, end-to-end customized single-domain antibody (sdAb/VHH) pre-discovery CRO services to support innovative pharmaceutical companies in their drug discovery programs.
AlpVHHs offers full-service VHH discovery and engineering, supporting your compound pipeline from antigen design to humanization and affinity maturation.
Our expert R&D team has over 10 years of experience in VHH field. We have collaborated with more than 300 biotech and biopharma companies, as well as universities, successfully delivering over 1,000 sdAb pre-discovery projects. Additionally, more than 20 collaborative projects have successfully progressed to IND approval.
If you are looking for tailored nanobody (sdAb/VHH) discovery and development solutions, AlpVHHs offers comprehensive single-domain antibody pre-discovery CRO services to support your project from start to finish. Learn more about our services here.
