Yeast surface display (YSD) is a "whole-cell" platform for heterologous expression of proteins immobilized on the surface of yeast cells. YSD combines the advantages offered by eukaryotic systems, such as post-translational modifications, proper folding and glycosylation of proteins, ease of cell culture and genetic manipulation, and allows for protein immobilization and recovery. In addition, proteins displayed on the surface of yeast cells may show increased stability to changes in temperature, pH, organic solvents, and proteases. The platform has been used to study protein-protein interactions, antibody design, and protein engineering. Other applications of YSD include library screening, whole proteome studies, bioremediation, vaccine and antibiotic development, biosensor production, ethanol production, and biocatalysis.

The efficacy of yeast surface display in protein engineering was first validated for affinity maturation of existing protein binding scaffolds, and subsequently it has been shown to be very useful for the isolation of new protein binders from combinatorial libraries. Combinatorial libraries of a variety of immunoglobulin and non-immunoglobulin scaffolds have been generated to enhance existing affinities or introduce de novo binding functionality to a wide range of targets, including small organic molecules, peptides, and soluble and membrane proteins. Single-chain or multi-chain immunoglobulin scaffolds include antibody single-chain variable fragments (scFv), antibody fragment antigen binding (Fab) parts, partial immunoglobulin G (IgG), whole IgG, camel single domain antibodies (VHH) and single-chain T cell receptors (scTCR).

I. Screening of Antibodies by Yeast Surface Display Technology

The target protein is linked by two epitope tags: a 9-amino acid hemagglutinin antigen (HA) tag and a 10-amino acid c-myc tag, and fused to the C-terminus of the a-lectin Aga2p subunit. Protein display on the surface of yeast cells. After translation, the 69-amino acid Aga2p subunit binds to the 725-amino acid a-lectin Aga1p subunit through two disulfide bonds. The fusion protein is then secreted into the extracellular space, where Aga1p is covalently anchored to the cell wall via β1,6-glucan. As a result, the protein of interest is displayed on the cell surface, where it is more accessible to soluble ligands. Functional display of the protein of interest (shown here as a scFv modified from PDB 1X9Q using UCSF Chimera packaging) can be detected by fluorescently labeled antibodies or ligands specific to the native fold (red star).

Yeast surface display technology is an efficient antibody screening method that fuses antibody genes with yeast surface proteins to display antibodies on the surface of yeast cells, thereby enabling specific binding to target antigens. The key advantages of this technology are the easy cultivation, high expression ability and good stability of yeast cells, which can quickly construct a diverse antibody library for high-throughput screening. Through binding to the target antigen, antibodies with strong affinity and high specificity can be screened. Common screening methods include yeast display affinity screening and flow cytometry (FACS). Compared with traditional mammalian cell screening methods, yeast surface display is not only low-cost, but also shortens the screening cycle, and has strong experimental operability and controllability. Therefore, it is widely used in fields such as antibody discovery, drug development and vaccine research, and is particularly important in the development of new therapeutic antibodies.

Figure 1 Schematic diagram of yeast surface display

II. Yeast Cell Surface Engineering and Its Application in Biotechnology

Current yeast display technology takes advantage of the GPI (glycosylphosphatidylinositol) anchor protein naturally present in the cell wall, which can serve as an anchor unit for displaying recombinant proteins (such as antibody fragments). In this setting, the protein of interest is used to the C- or N-terminal part of the respective anchor protein to guide the bound fusion protein to the extracellular surface. In recent years, several different anchor proteins have been identified that facilitate the display of foreign proteins on yeast cells, but the most prominent one is the Aga2p-dependent surface display invented by Boder and Wittrup in 1997. This system has previously been used to design various antibody formats, such as scFv fragments, Fab fragments, Fcabs, and llama single domain antibody VHH fragments, with the aim of isolating clones with improved performance.

III. Genetic Strategies for Improving Yeast Surface Display

1. Yeast Plasmids

Synthetic yeast plasmids are extrachromosomal genetic elements used to control the expression of heterologous proteins, designed to drive gene expression under the control of regulatory sequences (i.e., promoters, terminators, transcription factors, etc.). In addition, plasmid copy number can affect gene expression levels. YSD plasmid protein expression depends on promoter strength. Both constitutive and inducible promoters are used to display proteins. The most commonly used promoters are the galactose promoter (GAL1/GAL10) expressed in Saccharomyces cerevisiae, and the GAP and AOX1 promoters expressed in Pichia pastoris.

2. Signal Peptide Sequence

The anchor contains two main parts: (1) a signal peptide sequence (SS) that participates in protein transport through the protein secretion pathway; (2) an anchor fused to the POI. Modification of the SS has an important effect on the improvement of the production level of the displayed POI. In general, the use of natural signal peptides, i.e., a-lectin, SED1p, Pir1p, and Flo1p have been shown to have good protein expression levels using their own signal peptides.

3. Anchoring Proteins

The most common anchors are GPI-dependent CWPs (cell wall proteins), which provide a covalent bond between the target protein and the cell wall β-1,6 glucan. On the other hand, Pir-CWP promotes the covalent attachment of fusion proteins to cell wall β-1,3 glucan and structural proteins via disulfide bonds. The Aga1-Aga2 anchor, originally developed by Boder and Wittrup, has been used for the expression of several proteins.

4. Selection of Yeast Strains for Surface Display of Yeast

The genomic and metabolic background of the applied yeast strain is an important trait that affects the YSD of native or exogenous proteins. Synthetic biology approaches combined with genomic technologies such as CRISPR/Cas systems can develop yeast strains with fine-tuned heterologous protein expression. New yeast strains are designed by changing the cell wall composition or protein secretion pathway of yeast. The latter includes vesicle transport engineering and the development of platforms for the simultaneous secretion of soluble proteins and surface-displayed proteins to simplify their characterization.

TekBiotech focuses on providing customers with high-quality and cost-effective early discovery technology services for antibody drugs. We have 10 years of project development experience and insights in drug antibody discovery, and have accumulated sufficient experience in antibody customization and recombinant protein production. Based on the Yeast Display Technology platform, TekBiotech can provide customers with a series of monoclonal antibody preparation services including but not limited to rabbit, mouse, sheep, camel and other species, as well as a complete traceability document system.