In the field of nanobody (VHH) development, display technology is a core tool determining screening efficiency and success rate. Currently, the two most widely used platforms—phage display and yeast surface display—each have their own advantages and applicable scenarios.
Phage display, with its low equipment requirements and large library capacity, has become the mainstream choice for early-stage antibody screening; while yeast display, relying on its advantages in eukaryotic expression and flow cytometry sorting, excels in high-quality antibody screening and functional screening.
AlpVHHs have established mature platforms for both:
● Phage display technology platform
● Yeast display technology platform
We will systematically compare the two technologies from both theoretical and application perspectives to help you choose the optimal R&D strategy based on your project needs.
Yeast display technology is a widely used tool in research and industrial production. It was first reported by Maarten P. Schreuder and colleagues in 1993 on the display of exogenous proteins from the yeast cell wall, and K. Dane Wittrup and colleagues reported the construction and screening of yeast surface peptide display libraries in 1997. Currently, yeast surface display technology is widely used in protein engineering, antibody development, affinity modification, and other fields.
The cell wall of Saccharomyces cerevisiae is thick and tough, about 200 nm, located outside the plasma membrane. It is mainly composed of mannoprotein and β-glucan and has a bilayer structure, including an inner glucan backbone layer and a fibrous or brush-like outer layer mainly composed of mannoprotein (Figure 1).
Fig 1. Cell surface structure of Saccharomyces cerevisiae
Typically, the display of a target protein is achieved through fusion expression with a host yeast cell wall protein. Commonly used anchoring proteins include α-lectin, Flo1p, Yps1p, Cwp2p, Sed1p, and Pir1-4 (Figure 2). The displayed protein is transported via the secretory pathway, first to the endoplasmic reticulum lumen, then from the endoplasmic reticulum to the Golgi apparatus, and finally anchored to the cell wall surface. Commonly used anchoring proteins, such as α-lectin, consist of a core subunit encoded by the Aga1 gene and a core subunit encoded by the Aga2 gene. The Aga1 and Aga2 subunits are linked by a disulfide bond and anchored to the cell wall via the glycosylphosphatidylinositol (GPI) at the C-terminus of the AGA1 protein. The target protein can be constructed by fusing it to either the N-terminus or C-terminus of the Aga2 subunit. The nanobody fragment VHH, fused to either the N-terminus or C-terminus, can be efficiently expressed and screened.
Figure 2. Different Saccharomyces cerevisiae cell surface display systems
Yeast surface display is mainly screened using MACS/FACS, with the screening process monitored using FACS, which is very intuitive and convenient.
Phage display is a molecular biology technique that utilizes laboratory evolution to screen for peptides or antibodies with specific binding abilities in a high-throughput, high-content manner. It has been widely used in basic and applied research related to biomedicine, new materials, new energy, and environmental protection. This technique was initially developed by George P. Smith in the mid-1980s and applied to antibody engineering by John McCafferty and Gregory Winter in the early 1990s. In 2018, two pioneers of phage display research, American scientist George P. Smith and British scientist Gregory P. Winter, were awarded the Nobel Prize in Chemistry.
Phage display technology involves inserting exogenous peptides, antibodies, or other fragments into the structural genes of a phage, commonly through fusion with PIII or PVIII. The molecules displayed on the phage surface retain their biological activity. To select desired molecules from the phage library, a "screening" process is required. Simply put, the peptide or protein library displayed on the phage surface must specifically recognize and bind to the target antigen. After sufficient incubation, weakly bound or unbound free phages are washed away. The specifically bound target phages are then eluted, infecting *E. coli*, and amplified to obtain the next round of daughter phage libraries. After 2-3 rounds of "adsorption-elution-amplification" enrichment, the proportion of phages specifically binding to the antigen gradually increases. Finally, peptides or proteins capable of recognizing the target molecule are obtained and can be used for subsequent experiments.
Although both display systems can achieve genotype and phenotype unification and are widely used in antibody development, they differ significantly in terms of protein expression systems, library capacity, and screening methods.
Phage Display | VS | Yeast Surface Display |
Prokaryotic | System | Eukaryotic |
One copy | Display level | 10^4~10^5 Copies |
10% | Display percent | 65%~80% |
VHH,ScFv,peptide,Fab etc. | Antibody Format | VHH,ScFv,peptide,Fab, IgG etc. |
Limited/biased | Display diversified | Excellent |
High | False positive | Low |
Solid / liquid phase | Panning | MACS & FACS |
Protein, cell etc. | Panning antigen format | Protein |
<1000 (Sanger) | Throughput | >10,0000 (NGS) |
BCMA, CLDN18.2, Trop2, GPRC5D, MSLN etc. | Representive Case | BAFFR, PD-L1, IGF-1R, CD16a, HSA,TfR1 etc. |
Bacteriophages are viruses that require infection with *E. coli* to replicate and amplify, making them a prokaryotic expression system. However, antibodies originate from eukaryotes, and due to codon bias, some antibodies cannot be effectively displayed or may become toxic, leading to clone loss. Yeast, on the other hand, is a eukaryotic expression system. The displayed antibodies are first transported to the endoplasmic reticulum lumen and then processed by the Golgi apparatus, ensuring correct protein folding and exhibiting modification characteristics more closely resembling those of eukaryotic expressed proteins, as well as better biological activity.
Because the transformation efficiency of *E. coli* is much higher than that of yeast cells, phage libraries can generally reach 10⁹. For a long time, due to technological limitations, the library capacity of yeast libraries could only reach 10⁶–10⁷. Therefore, yeast surface display was generally used for affinity maturation, and for such mutant libraries, the library capacity requirement was not high. Nanobodies do not involve in vitro recombination of the light and heavy chains of traditional antibodies. AlpVHHs through technological improvements, can currently guarantee a library capacity of 10⁸, which fully meets the needs of nanobodies research and development.
Phage display libraries are constructed using either enzyme digestion and ligation or homologous recombination. Enzyme digestion and ligation is the most commonly used method due to its lower cost compared to in vitro homologous recombination. In vitro homologous recombination can significantly improve ligation efficiency, but it is very expensive. Yeast surface display involves electroporating competent yeast cells with a linearized target fragment and a linearized vector in a specific ratio. The fragment and vector then circularize into a plasmid within the yeast cells through homologous recombination. This method is inexpensive and simple to perform.
In antibody development, phage display libraries typically use monovalent display. To achieve monovalent display, the display efficiency after packaging is approximately 10%. However, there is no effective detection method to monitor the packaging efficiency of the library, which is very troublesome. Poor screening may be due to poor display efficiency, introducing more variables. It is recommended to use anti-g3p antibodies to detect packaging efficiency.
Yeast surface display carries detection tags such as HA, Flag, and myc, which makes it very convenient to determine the library expression efficiency via flow cytometry. The display efficiency can generally reach 60% to 80%. Yeast surface display is multivalent display, with approximately 104 to 105 copies on a single yeast cell.
Phage display has a limited molecular weight capacity. Generally, peptides, VHH, ScFv, or Fab fragments are fused to the PIII protein. Fab fragments are relatively large for phages. Large molecular weights can affect phage infection and packaging. However, yeast display, in addition to displaying the aforementioned fragments, can also display the entire full-length antibody IgG molecule, which phage display cannot do.
Phage display systems exhibit significant bias, which mainly arises from three factors:
● Since bacteriophages operate in a prokaryotic system while antibodies originate from eukaryotes, differences in codon usage preferences and the potential toxicity of certain expressed molecules to E. coli can lead to unequal growth rates among clones during propagation, and may even result in the loss of some clones;
● Clones lacking an inserted fragment grow much faster than those containing inserts, thereby reducing the overall quality of the library;
● Incorrect open reading frames (ORFs) can also cause certain clones to grow more rapidly than others.
In contrast, yeast systems generally do not exhibit these issues, as growth tends to be more uniform.
A key advantage of yeast display lies in the precise control of selection parameters during FACS (fluorescence-activated cell sorting). The percentage of the collected population, signal normalization, and the desired binding affinity can all be defined by setting appropriate gates in flow cytometry. This ability to explicitly define binding criteria during selection provides a significant advantage over phage display platforms, where variant identification relies on washing steps rather than real-time kinetic observation. In other words, the systematic bias toward desired binding characteristics that can be introduced during yeast library selection is not achievable in phage display screening.
Yeast surface display is highly compatible with the quantitative and multiparametric analysis enabled by flow cytometry. Experimental conditions—including buffer composition, pH, temperature, and antigen concentration—can be precisely controlled. Clones that bind to the target protein under specific incubation conditions can be selectively enriched, such as those exhibiting improved stability under defined conditions. Furthermore, each individual cell in the library can be analyzed in real time for its display level and antigen-binding capability, allowing for the isolation of specific cell populations.
Below, we present a practical example to illustrate the advantages of yeast-based screening:
a. Affinity-Based Sorting
In phage display, high-affinity clones are typically enriched by increasing selection pressure, such as raising the coating antigen concentration or strengthening washing conditions. However, these processes cannot be effectively monitored in real time. Moreover, the selected clones can only be classified as positive or negative, without distinguishing differences in binding affinity. As a result, affinity comparisons must be performed after protein expression, which significantly increases workload and cost.
In contrast, yeast display enables affinity-based discrimination during the screening process itself. Libraries are incubated with target proteins at defined concentrations, allowing antibody-displaying clones with different affinities to be distinguished under specific antigen concentrations and sorted accordingly. As illustrated in Figure 6, flow cytometry profiles of cells incubated with varying concentrations of target protein show clear separation of cell populations with different affinities at 11.1 nM. These populations can be gated into high-, medium-, and low-affinity groups, enabling direct isolation of high-affinity clones.
This approach also provides insight into whether high-affinity populations exist within the library and reveals the distribution of clones across different affinity ranges.
Fig 3. Flow cytometry sorting based on different affinities (target protein incubation concentrations from left to right are 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, and 1.2 nM).
b. Blocking of Active Antibody Screening
For immune checkpoint targets, obtaining antibodies with functional activity requires the ability to block receptor-ligand interactions. In conventional phage display workflows, binding antibodies are first isolated, followed by expression and subsequent validation to determine whether they can block receptor-ligand binding. This process is inefficient and often yields a relatively low success rate.
In contrast, yeast surface display enables direct selection of blocking antibodies through competitive sorting. This is achieved by fluorescently labeling the ligand protein or a benchmark antibody and performing competition-based screening. For example, in the selection of PD-L1 blocking antibodies, PD-L1 and PD-1 can be labeled with different fluorophores and incubated simultaneously with the yeast library. Clones are then sorted from the population that show positive PD-L1 binding signals but negative PD-1 binding signals.
As illustrated in Figure 4, cells gated in the F-Q1 region are enriched. Upon further characterization, these clones bind to PD-L1 but no longer interact with PD-1, indicating that the antibodies effectively block the PD-L1/PD-1 interaction. This approach enables highly efficient identification of functional blocking antibodies.
(a) (b)
Fig 4. Sorting and identification chromatograms of specific epitopes
(a: PD-L1 labeled fluorescent 650, PD-1 labeled fluorescent PE, sorting region F-Q1 with positive 650 signal and negative PE signal;
b: flow cytometry identification chromatogram of sorted products, left signal PD-L1-647/HA-488, right signal PD-1-PE/PD-L1-647).
Phage screening is similar to ELISA, typically involving 2-3 rounds of panning. To ensure clonal diversity, the number of panning rounds should be minimized to avoid excessively high output positivity rates. Secondly, filamentous phages are relatively sticky and prone to nonspecificity, while yeast screening controls nonspecificity by detecting a double-positive signal from the binding of the expression tag and antigen. Yeast display generally involves MACS enrichment in the first round and FACS sorting in the second round, directly sorting specific populations. By controlling the sorting precision, a positivity rate of over 90% can be achieved without losing diversity. Traditional clonal analysis involves selecting single clones for validation and Sanger sequencing, typically selecting 5-10 plates. This method often selects clones with high abundance, making it difficult to obtain low-abundance clones, resulting in limited data. Besides selecting single clones, yeast display also employs a critical point method of sorting over 100,000 positive clones from a specific population for NGS sequencing. The depth of analysis is unmatched by traditional clone selection. The obtained data can be used for abundance ranking or clustering based on CDR3, easily yielding hundreds of candidate clones. Phages, due to their stickiness and tendency to be nonspecific, result in very low confidence levels for positive clones in NGS sequencing; often, only a few of the synthesized clones are positive.
● Initial screening of large library
● Rapid acquisition of candidate molecules
● Cost-sensitive projects
Recommended Platform: AlpVHHs Phage Display Technology Platform
● Large library size up to 10^9 CFUs.
● Absolutely background free cloning system.
● Available for antigen presented on the surface of whole cells or tissue.
● Affinity maturation
● Screening of functional antibodies (e.g., blocking antibodies)
● Screening of high-quality candidate molecules
● Fine optimization of nanobodies
Recommended Platform: AlpVHHs Yeast Display Technology Platform
● Stable platform with extensive experience.
● Expression efficiency ranging from 70% to 80%.
● High-quality yeast display library, with a capacity of up to 10^8 CFUs.
In practical projects, we recommend combining phage display with yeast display to fully leverage the advantages of both platforms, achieving an optimal balance between efficiency and quality.
Phage Display → Large-scale initial screening
Yeast Display → Precise screening and functional validation
By adopting these combined strategies, you can simultaneously address:
✔ Library Diversity
✔ Screening Accuracy
✔ Functional Validation
It is important to emphasize that these two platforms are not mutually exclusive; rather, they are highly complementary:
● Phage Display: Efficiently solves the "existence" problem—rapidly obtaining candidate molecules from massive libraries
● Yeast Display: Precisely answers the "goodness" problem—achieving fine-grained screening based on affinity, specificity, and function
However, the true determinant of a project's success lies not only in whether a combined strategy is adopted, but also in the capabilities of the platforms themselves and the depth of their integration.
In the AlpVHHs yeast display technology platform, we optimize yeast expression systems and flow cytometry to achieve high-precision, multi-dimensional screening, effectively obtaining molecules with high affinity and high stability. Simultaneously, combined with a mature phage display system, we support the rapid and efficient enrichment of candidate molecules from ultra-large-scale libraries. Based on these integrated platforms, we have:
✔Successfully delivered 1,000+ VHH discovery projects
✔Advanced 20+ projects into the IND stage
✔Builded an integrated capability system covering library construction→screening→validation→optimization
Are you looking for a reliable nanobody development partner? Contact us today to learn more about our Custom VHH Discovery & Development service and accelerate your R&D process together.
