Eukaryotic System-Based Targeted Drug Discovery Platform: Yeast Display Technology-Yeast Display Technology Special Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Eukaryotic System-Based Targeted Drug Discovery Platform: Yeast Display Technology

Yeast display technology (YSD) enables the display of exogenous proteins on the cell surface through fusion with anchor proteins. Compared to previously established phage and bacterial display systems, the yeast expression system offers distinct advantages. First, yeast cells are large, easy to culture, and edible, making them widely used in the food industry due to their safety. Second, as a eukaryotic single-celled organism, yeast possesses a well-established and convenient genetic manipulation system. Its post-translational modification capabilities promote the correct folding and secretion of large or complex proteins, ensuring accurate protein conformation. Finally, the primary advantage of the yeast system is its compatibility with fluorescence-activated cell sorting (FACS). This allows direct assessment of displayed protein specificity without the need for complex soluble protein expression or purification processes.

This technology is widely used in protein engineering, antibody development, and affinity maturation. Applications include:1.Antigen Display: ①Oral vaccine development (e.g., displaying H7N9 influenza virus antigen on the yeast surface while maintaining intact antigen structure).②Epitope mapping (displaying antigen truncations or mutants on the yeast surface combined with flow cytometry to identify the amino acid sequences on the antigen that bind to corresponding antibodies).2.Antibody Display:①Antibody screening (directly sorting antibody molecules with blocking activity through competitive binding assays of displayed antibodies).②Antibody affinity maturation (constructing antibody mutant libraries and using FACS to screen for high-affinity mutants).③Antibody humanization (transferring CDR regions from non-human antibodies onto human antibody frameworks and displaying the humanized antibodies on yeast).④Bispecific antibodies (co-expressing two antibodies to simultaneously target two different antigens, exerting dual-target killing effects on cancer cells).

Additionally, the technology can be used to display enzymes, serving as whole-cell catalysts to improve biological efficiency or synthesize sweeteners. In agricultural and food applications, it is used to develop biocontrol agents, such as expressing flagellin to increase tomato resistance to bacterial infection.

Different yeast surface display systems depend on the genetic background and metabolic characteristics of each host. Since the discovery of the yeast surface display system by Boder and Wittrup in 1997, Saccharomyces cerevisiae has been the most common and ideal host. Among these, the S. cerevisiae α-agglutinin system is the most popular. The yeast cell wall has a sandwich-like structure: mannan (outer layer) - protein (intermediate layer) - glucan (inner layer). This structure provides the basis for exogenous protein display.

Display Mechanism: The exogenous protein is linked to the host's anchor protein (composed of Aga2p and Aga1p) and displayed on the cell surface. As shown in Figure 1, the exogenous target protein (blue) is fused to the C-terminus or N-terminus of Aga2p. Aga2p is linked via disulfide bonds to the scaffold subunit Aga1p, which is anchored to the glucan layer. The fusion protein then enters the ER-Golgi secretory pathway, where disulfide bond formation and glycosylation are completed, before being transported to the cell surface. Surface expression can be detected using fluorescently labeled antibodies against tags such as HA or Myc. Nanobodies are generally fused to the N-terminus of Aga2p, while scFvs, Fabs, and intact IgG? are linked to the C-terminus. Fusion at either the N-terminus or C-terminus does not disrupt protein structure or affect surface display efficiency.

However, the yeast display system also has drawbacks. Compared to phage display systems, its library capacity is smaller (10? - 10?). Nevertheless, its manipulable eukaryotic genetic characteristics enable diverse applications, and it will play a significant role in the future.

Figure 1: Saccharomyces cerevisiae* surface α-agglutinin display system [1].

References

[1] Uchański T, Z?gg T, Yin J, et al. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep. 2019;9(1):382.

Previous:Common Problems and Solutions for Yeast Two-hybrid Experiments Next:None!