Yeast Display System Research Articles

Abstract 41: Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning

Abstract Purpose: Mesothelin (MSLN) is a surface antigen associated with tumor invasion and is overexpressed in mesothelioma and lung, pancreatic, ovarian, and other cancers. Chimeric antigen receptor (CAR) T cells targeting MSLN have been tested in patients with MSLN-positive solid tumors. Despite reported patient fatalities in trials sponsored by PENN, Memorial Sloan Kettering/Atara Bio, and MD Anderson/TCR2, the design approach for MSLN CAR remains constructing high affinity CARs to maximize target cell killing. In this study, we designed MSLN CARs with two different antibodies: one targeting human MSLN specifically and another binding both human and mouse MLSN with comparable affinities. The objectives are to 1) elucidate the risk of fatal on-target off-tumor toxicity associated with high affinity MSLN CAR, and 2) demonstrate how optimizing the affinity of CARs can render CAR T cells to spare normal tissues while retaining cytotoxicity against tumors. Methods: To asses on-target off-tumor toxicity in animal models, we developed a CAR using a nanobody isolated from an immune alpaca library with nanomolar affinity for both human and mouse MSLN (labeled as hmMSLN CAR). For comparison, we constructed a CAR using a scFv specific only to human MSLN, designated hMSLN CAR. Each CAR is designed to co-express somatostatin receptor 2 to enable PET/CT imaging of CAR T cells with 18F-NOTA-Octreotide. Anti-tumor activities and toxicities were assessed in NSG mice using MSTO-211H cell line expressing human MSLN. Results: In NSG mice bearing subcutaneous MSTO-211H tumors, hMSLN CAR T cells eliminated tumors rapidly without inducing any overt toxicity. In contrast, hmMSLN CAR T cells not only failed to control tumor growth but also caused severe toxicity, resulting in significant body weight loss and necessitating euthanasia. PET/CT imaging revealed while hMSLN CAR T cells expanded exclusively within the tumor and subsequently contracted after tumor elimination, hmMSLN CAR T cells exhibited extensive infiltration and expansion in the lungs, liver, and bone marrow, indicating off-tumor, normal tissue-driven proliferation of CAR T cells. To test if affinity-tuned hmMSLN CAR T cells can selectively target tumors, we used a yeast display system to screen for affinity variants of the hmMSLN CAR. We identified one micromolar affinity CAR that binds approximately 1,000 times weaker to MSLN than the parental CAR. In stark contrast to the onset of severe toxicity soon after the administration of the parental hmMSLN CAR T cells, animals treated with micromolar affinity hmMSLN CAR T cells exhibited tumor regression without overt toxicity. Conclusions: Our findings underscore the risks of using high affinity CAR against MSLN, which may cause unpredictable on-target, off-tumor toxicities. Fine tuning the affinity of MSLN CAR offers a promising strategy for developing a potent and safe CAR for the treatment of MSLN-expressing solid tumors. Citation Format: Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Ju Y. Son, Niloufar Salehi, Irene M. Min, Moonsoo M. Jin. Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 41.

Competitive blocking of LRP4-sclerostin binding interface strongly promotes bone anabolic functions.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

Evaluation of the yeast surface display system for screening of functional nanobodies

Yeast surface display is a powerful technology used to isolate and engineer proteins to improve their activity, specificity, and stability. In this method, gene expression is regulated by promoters, and secretion efficiency is affected by secretion signals. Furthermore, both the accessibility and activity of the displayed proteins are affected by the length of anchor proteins. The ideal promoter, secretion signal, and anchor protein combination depend on the proteins of interest. In this study, we optimized a yeast surface display suitable for nanobody evaluation. We designed five display systems that used different combinations of promoters, secretion signals, and anchor proteins. Anti-hen egg-white lysozyme nanobody was used as the model nanobody. The amount of nanobodies displayed on yeast cells, the number of antigens bound to the displayed nanobodies, and the display efficiency were quantified. Overall, we improved the yeast display system for nanobody engineering and proposed its optimization.

An overview of yeast cell wall proteins and their contribution in yeast display system

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Although, recent advances have greatly expanded the capability of yeast surface display, the protein display system is still far away from industrial application. One of the major components of a stable, efficient and successful yeast surface display system is cell wall anchor protein with which our desired foreign protein will be attached. We studied 80 different yeast cell wall anchored proteins originated mostly from Saccharomyces cerevisiae and Candida albicans. We studied in details all the cell wall proteins in order to find out suitable cell wall proteins to recommend for the researchers to use in the construction of yeast display system. We considered selective physical properties of different yeast cell wall proteins that are crucial for selecting best suited cell surface anchor proteins which are molecular weight, binding domain of anchor protein, length of amino acid and fusion site. Finally, our studies showed that Ccw11, Ccw12. Cwp1, Cwp2, Dan1, Gas1, Gas5, Exg1, Ycr89, Ecm33, Pga4, Sap9, Sap10, Pst1, Pir1, Pir2, Pir3, Pir4, Cis1, Scw4, Scw6, Bgl2, Uth1, Scw1 are the promising and suitable cell wall anchor proteins could be used in construction of yeast cell surface display system. Additionally, this review presents detailed information about all the cell wall proteins in a single work. The future researchers in this field will be able to construct more efficient yeast display system for recombinant protein production at industrial scale using the knowledge presented in this work.
 Asian J. Med. Biol. Res. June 2019, 5(4): 246-257

Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping.

Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.

An improved yeast surface display platform for the screening of nanobody immune libraries

Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.

Development of a modified yeast display system for screening antigen-specific variable lymphocyte receptor B in hagfish (Eptatretus burgeri)

The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS). We also demonstrated a strong specificity of the antigen-specific VLRBs, when expressed as a secreted protein using a mammalian expression system. Together, our findings suggest that the pYD8 vector system could be useful for screening antigen-specific hagfish VLRBs, and the specificity of secreted VLRB may have potential for a variety of biological applications.

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OBJECTIVES/SPECIFIC AIMS: The off-target and organ-specific toxicities observed in cancer immunotherapy present an obstacle to T-cell-based therapeutics. A recent clinical trial underscored the need for improved methods to define TCR specificity after melanoma patients treated with TCR engineered T-cells suffered from fatal cardiovascular toxicity arising from the unpredicted recognition of a muscle-specific peptide. METHODS/STUDY POPULATION: To address this drawback to T-cell-based immunotherapies, we developed a novel protein engineering approach to define the peptide specificity of a given TCR. Here, directed evolution in a yeast display system produced a large scale peptide library, where recognition by the melanoma reactive DMF5 TCR acted as the guiding selective pressure. After this technique identified a panel of putative cross reactive peptides, sequence analysis and computational modeling followed by kinetic binding experiments and structural analysis determined the DMF5 TCR recognizes 2 distinct classes of peptides through chemically distinct mechanisms. RESULTS/ANTICIPATED RESULTS: This information led to the rational, structure-based design of additional cross reactive peptides and introduced a unique approach to screen the human proteome and identify the TCR targets which triggered undesired autoimmunity when this molecule was used in clinical trials. DISCUSSION/SIGNIFICANCE OF IMPACT: The distinct chemical nature of the 2 peptide classes suggest TCRs are more cross reactive than previously thought, presenting an obstacle to cell-based immunotherapy. Defining the peptide specificity of TCRs is of high interest to the immunology community, and will lead to improved approaches to designing engineered TCRs for cell therapy.

猪瘟病毒E2蛋白BC抗原域的酿酒酵母表面展示

为构建猪瘟病毒(CSFV)E2蛋白的B、C抗原域(E2BC)酿酒酵母表面展示体系，以pVEXE2为模板，用PCR克隆了E2BC基因，并插入酵母展示表达载体p1v5AG的BamH I和EcoRI位点，构建重组酵母展示表达载体，用LiAC方法转化酿酒酵母细胞W303，构建重组酵母菌。重组菌经培养后，对酵母菌细胞进行针对His标签的间接免疫荧光染色，检测His-E2BC融合表达蛋白。结果显示，重组酵母菌表面有绿色荧光，表明E2BC蛋白成功表达于酵母表面，为开发酵母载体口服疫苗奠定了基础。 To construct the yeast display system of classical swine fever virus E2 glycoprotein BC antigenic domain, gene fragment was cloned by PCR using pVEXE2 as a template. E2BC was inserted into the site of BamH I and EcoR I of p1v5AG to produce recombinant surface displaying vector of p1E2BCAG. p1E2BCAG was transformed into Saccharomyces cerevisiae by LiAC method. After recombinant cells were cultivated, indirect immunofluorescence was used to detect E2BC expression. The results indicate that E2BC protein was displayed on the surface of Saccharomyces cerevisiae cell successfully. This work provides a foundation for the development of Saccharomyces cerevisiae vector oral vaccine of CSFV E2BC protein.

Combining Yeast Display and Competitive FACS to Select Rare Hapten-Specific Clones from Recombinant Antibody Libraries.

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 106) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene–protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.

Abstract A088: Increase lymphocyte infiltration overcomes tumor resistance to checkpoint blockade

Abstract Spontaneous T cell-inflamed tumor microenvironment is associated with clinical responses to immunotherapies, including immune checkpoint blockades. However, majority of established tumors often inhibit peripheral T cells entering tumor tissues for tumor destruction and/or prevent effective T cell priming. Previous studies in our lab have shown that upregulation of the tumor necrosis factor superfamily member LIGHT (also known as TNFSF14) in peripheral tissues results in T cell activation and migration into non-lymphoid tissues and formation of lymphoid-like structures, which lead to rapid T cell-mediated tissue destruction. Furthermore, local expression of LIGHT by tumor cells may promote the formation of lymphoid-like structures for direct T-cell sequestration and activation, leading to tumor regression. Those data suggest LIGHT as an interesting candidate to create T cell-inflamed tumor environment. To better evaluate the therapeutic efficacy of human LIGHT protein in both murine and human systems, we engineered human LIGHT by random mutation using yeast-display system, and were able to generate a human LIGHT mutant which binds to both murine and human receptors while remains stable. To specifically target LIGHT to tumor tissues, an antibody-LIGHT fusion protein was generated. In different mouse models, we found that antibody-LIGHT induces a T cell inflamed tumor environment. This is correlated with an upregulation of inflammatory cytokines and adhesion molecules, and leads to the rejection of established tumors. More importantly, we observed that LIGHT-mediated T cell inflamed environment is critical for restoring the efficacy of checkpoint blockade therapy. When we blocked lymphocyte trafficking, a tumor previously responding to checkpoint blockade became resistant to therapy. This process can be reversed by treating with LIGHT. Our study indicates that targeting LIGHT may be an effective strategy for cancer immunotherapy, especially those who do not response to checkpoint blockade due to the lack of infiltration. Citation Format: Haidong Tang, Yang Wang, Yang-Xin Fu. Increase lymphocyte infiltration overcomes tumor resistance to checkpoint blockade. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A088.

Directed evolution of a yeast-displayed HIV-1 SOSIP gp140 spike protein toward improved expression and affinity for conformational antibodies.

Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution.

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform.

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g., a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g., T cell activation by as few as 1-3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with K D values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display.

Engineered yeast for enhanced CO2 mineralization.

In this work, a biologically catalyzed CO2 mineralization process for the capture of CO2 from point sources was designed, constructed at a laboratory scale, and, using standard chemical process scale-up protocols, was modeled and evaluated at an industrial scale. A yeast display system in Saccharomyces cerevisae was used to screen several carbonic anhydrase isoforms and mineralization peptides for their impact on CO2 hydration, CaCO3 mineralization, and particle settling rate. Enhanced rates for each of these steps in the CaCO3 mineralization process were confirmed using quantitative techniques in lab-scale measurements. The effect of these enhanced rates on the CO2 capture cost in an industrial scale CO2 mineralization process using coal fly ash as the CaO source was evaluated. The model predicts a process using bCA2- yeast and fly ash is ~10% more cost effective per ton of CO2 captured than a process with no biological molecules, a savings not realized by wild-type yeast and high-temperature stable recombinant CA2 alone or in combination. The levelized cost of electricity for a power plant using this process was calculated and scenarios in which this process compares favorably to CO2 capture by MEA absorption process are presented.

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.

Development of Yeast Molecular Display Systems Focused on Therapeutic Proteins, Enzymes, and Foods: Functional Analysis of Proteins and its Application to Bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.

Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters

A novel adapter-directed yeast display system with modular features was developed. This display system consists of two modules, a display vector and a helper vector, and is capable of displaying proteins of interest on the surface of Saccharomyces cerevisiae through the interaction of two small adapters that are expressed from the display and helper vectors. In this report, an anti-VEGF scFv antibody gene was cloned into the display vector and introduced alone into yeast S. cerevisiae cells. This led to the expression and secretion of a scFv antibody that was fused in-frame with the coiled-coil adapter GR1. For display purposes, a helper vector was constructed to express the second coiled-coil adapter GR2 that was fused with the outer wall protein Cwp2, and this was genetically integrated into the yeast genome. Co-expression of the scFv–GR1 and GR2–Cwp2 fusions in the yeast cells resulted in the functional display of anti-VEGF scFv antibodies on the yeast cell surfaces through pairwise interaction between the GR1 and GR2 adapters. Visualization of the co-localization of GR1 and GR2 on the cell surfaces confirmed the adapter-directed display mechanism. When the adapter-directed phage and yeast display modules are combined, it is possible to expand the adapter-directed display to a novel cross-species display that can shuttle between phage and yeast systems.

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption

Vector engineering and gene disruption in host cells were attempted for the enhancement of alpha-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.