The Experts below are selected from a list of 12621 Experts worldwide ranked by ideXlab platform
Michael E. Birnbaum - One of the best experts on this subject based on the ideXlab platform.
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extracting and interpreting the effects of higher order sequence features on peptide mhc binding
bioRxiv, 2020Co-Authors: Zheng Dai, Michael E. Birnbaum, Brooke D Huisman, David K GiffordAbstract:Understanding the factors contributing to peptide MHC (pMHC) affinity is critical for the study of immune responses and the development of novel therapeutics. Developments in Yeast Display platforms have enabled the collection of pMHC binding data for vast libraries of peptides. However, methods for interpreting this data are still at an early stage. In this work we propose an approach for extracting peptide sequence features that affect pMHC binding from such datasets. In the process we develop the theoretical framework for fitting and interpreting these features. We demonstrate that these features accurately capture the kinetics underlying pMHC binding, and can be used to predict pMHC binding well enough to rival the current state of the art. We then analyze the extracted factors and show that they correlate with our current structural understanding of MHC molecules. Finally, we discuss the implication these factors have on the complexity of peptide engineering.
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repertoire scale determination of class ii mhc peptide binding via Yeast Display improves antigen prediction
Nature Communications, 2020Co-Authors: Garrett C Rappazzo, Michael E. Birnbaum, Brooke D HuismanAbstract:CD4+ helper T cells contribute important functions to the immune response during pathogen infection and tumor formation by recognizing antigenic peptides presented by class II major histocompatibility complexes (MHC-II). While many computational algorithms for predicting peptide binding to MHC-II proteins have been reported, their performance varies greatly. Here we present a Yeast-Display-based platform that allows the identification of over an order of magnitude more unique MHC-II binders than comparable approaches. These peptides contain previously identified motifs, but also reveal new motifs that are validated by in vitro binding assays. Training of prediction algorithms with Yeast-Display library data improves the prediction of peptide-binding affinity and the identification of pathogen-associated and tumor-associated peptides. In summary, our Yeast-Display-based platform yields high-quality MHC-II-binding peptide datasets that can be used to improve the accuracy of MHC-II binding prediction algorithms, and potentially enhance our understanding of CD4+ T cell recognition. Identifying peptides that can bind major histocompatibility complex II (MHC-II) is important for our understanding of T cell immunity and specificity. Here the authors present a Yeast-Display library screening approach that identifies more potential binders than various reported algorithms to help expand our understanding for antigen presentation.
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repertoire scale determination of class ii mhc peptide binding via Yeast Display improves antigen prediction
Nature Communications, 2020Co-Authors: Garrett C Rappazzo, Michael E. Birnbaum, Brooke D HuismanAbstract:CD4+ helper T cells contribute important functions to the immune response during pathogen infection and tumor formation by recognizing antigenic peptides presented by class II major histocompatibility complexes (MHC-II). While many computational algorithms for predicting peptide binding to MHC-II proteins have been reported, their performance varies greatly. Here we present a Yeast-Display-based platform that allows the identification of over an order of magnitude more unique MHC-II binders than comparable approaches. These peptides contain previously identified motifs, but also reveal new motifs that are validated by in vitro binding assays. Training of prediction algorithms with Yeast-Display library data improves the prediction of peptide-binding affinity and the identification of pathogen-associated and tumor-associated peptides. In summary, our Yeast-Display-based platform yields high-quality MHC-II-binding peptide datasets that can be used to improve the accuracy of MHC-II binding prediction algorithms, and potentially enhance our understanding of CD4+ T cell recognition.
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Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity.
eLife, 2020Co-Authors: Juan L. Mendoza, Suzanne Fischer, Marvin H. Gee, Lilian H. Lam, Simon Brackenridge, Fiona Powrie, Michael E. Birnbaum, Andrew J. Mcmichael, K. Christopher Garcia, Geraldine M. GillespieAbstract:T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I Yeast Display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.
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antigen identification for orphan t cell receptors expressed on tumor infiltrating lymphocytes
Cell, 2017Co-Authors: Marvin H. Gee, Juan L. Mendoza, Suzanne Fischer, Michael E. Birnbaum, Arnold Han, Shane Lofgren, John F Beausang, Michael T Bethune, Xinbo Yang, Raquel GomezeerlandAbstract:The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used Yeast-Display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of “orphan” T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile identification of tumor antigens through unbiased screening.
David M Kranz - One of the best experts on this subject based on the ideXlab platform.
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t cell receptor engineering and analysis using the Yeast Display platform
Methods of Molecular Biology, 2015Co-Authors: Sheena N Smith, Daniel T Harris, David M KranzAbstract: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.
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structural features of t cell receptor variable regions that enhance domain stability and enable expression as single chain vαvβ fragments
Molecular Immunology, 2009Co-Authors: Sarah A Richman, David L Donermeyer, Michelle L Dossett, Philip D Greenberg, Paul M Allen, David H Aggen, David M KranzAbstract:Abstract The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to Display on phage or Yeast in their wild-type form. However, through random mutagenesis and Yeast Display engineering, it has been possible to isolate scTCR mutants that are properly folded and Displayed on the Yeast surface. These Displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in Escherichia coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed Display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for Yeast surface Display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Vα–Vβ interface, the HV4 of Vβ, and the region of the Vα and Vβ domains normally apposed against the constant (C) domains. Consistent with the role of the V–C interface in surface Display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain Display and αβ chain association on the Yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the VαCα/VβCβ constructs. Therefore, we describe further optimization of the current strategy for surface Display of the single-chain format in order to facilitate Yeast Display engineering of a broader range of scTCRs.
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development of a novel strategy for engineering high affinity proteins by Yeast Display
Protein Engineering Design & Selection, 2006Co-Authors: Sarah A Richman, S J Healan, K S Weber, David L Donermeyer, Michelle L Dossett, Philip D Greenberg, Paul M Allen, David M KranzAbstract:Yeast Display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that Yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound Yeast. High-affinity T cell receptors (TCRs) Displayed on Yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of Yeast Displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As Yeast Display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.
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high affinity t cell receptors from Yeast Display libraries block t cell activation by superantigens
Journal of Molecular Biology, 2001Co-Authors: Michele C Kieke, Eric V Shusta, Dane K Wittrup, Eric J Sundberg, Roy A Mariuzza, David M KranzAbstract:The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of Yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.
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in vitro evolution of a t cell receptor with high affinity for peptide mhc
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Phillip D Holler, Eric V Shusta, David M Kranz, Dane K Wittrup, Philmore O Holman, Sean OherrinAbstract:T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using Yeast Display and selection from a library of Vα CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (KD ≈ 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3α that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.
Eric V Shusta - One of the best experts on this subject based on the ideXlab platform.
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a Yeast Display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes
Protein Engineering Design & Selection, 2019Co-Authors: Jason M Lajoie, Yong Ku Cho, Dustin C Frost, Samantha Bremner, Eric V ShustaAbstract:Yeast Display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune Yeast Display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional Yeast Display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.
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Yeast Display biopanning identifies human antibodies targeting glioblastoma stem-like cells
Scientific Reports, 2017Co-Authors: Michael Zorniak, Yong Ku Cho, Benjamin J. Umlauf, Paul A Clark, Eric V Shusta, John S. KuoAbstract:Glioblastoma stem-like cells (GSC) are hypothesized to evade current therapies and cause tumor recurrence, contributing to poor patient survival. Existing cell surface markers for GSC are developed from embryonic or neural stem cell systems; however, currently available GSC markers are suboptimal in sensitivity and specificity. We hypothesized that the GSC cell surface proteome could be mined with a Yeast Display antibody library to reveal novel immunophenotypes. We isolated an extensive collection of antibodies that were differentially selective for GSC. A single domain antibody VH-9.7 showed selectivity for five distinct patient-derived GSC lines and visualized orthotopic GBM xenografts in vivo after conjugation with a near-infrared dye. These findings demonstrate a previously unexplored high-throughput strategy for GSC-selective antibody discovery, to aid in GSC isolation, diagnostic imaging, and therapeutic targeting.
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Yeast Display-Based Antibody Affinity Maturation Using Detergent-Solubilized Cell Lysates
Yeast Surface Display, 2015Co-Authors: Benjamin J. Tillotson, Jason M Lajoie, Eric V ShustaAbstract:It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted Yeast Display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens. The following protocol details the incorporation of cell lysates into a kinetic screen designed to obtain antibodies with improved affinity via slowed dissociation from an MP antigen.
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facile chemical functionalization of proteins through intein linked Yeast Display
Bioconjugate Chemistry, 2013Co-Authors: Carrie J Marshall, Nitin Agarwal, Jeet Kalia, Vanessa A Grosskopf, Nicholas A Mcgrath, Nicholas L Abbott, Ronald T Raines, Eric V ShustaAbstract:Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the Yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release Yeast surface-Displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were Displayed on the Yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the Yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of Displayed proteins, but nonspecific reactions with other Yeast proteins were det...
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antibody affinity maturation using Yeast Display with detergent solubilized membrane proteins as antigen sources
Protein Engineering Design & Selection, 2013Co-Authors: Benjamin J. Tillotson, Inigo Fernandez De Larrinoa, Colin A Skinner, Derek M Klavas, Eric V ShustaAbstract:Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the Yeast surface Display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of Yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by Yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3-7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that Yeast Display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs.
Dimiter S Dimitrov - One of the best experts on this subject based on the ideXlab platform.
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engineered antibody ch2 domains binding to nucleolin isolation characterization and improvement of aggregation
Biochemical and Biophysical Research Communications, 2017Co-Authors: Rui Gong, Dimiter S Dimitrov, Jun Zheng, Xihai Chen, Qi ZhaoAbstract:Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a Yeast Display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this Yeast Display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on Yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264 and Leu309, in the β-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future.
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highly efficient selection of epitope specific antibody through competitive Yeast Display library sorting
mAbs, 2013Co-Authors: Vinita Puri, Emily Streaker, Zhongyu Zhu, Ponraj Prabakaran, Dimiter S DimitrovAbstract:Combinatory antibody library Display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a Yeast Display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a Yeast Display naive antibody library. All the Yeast Display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one ...
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Highly efficient selection of epitope specific antibody through competitive Yeast Display library sorting
mAbs, 2013Co-Authors: Vinita Puri, Emily Streaker, Zhongyu Zhu, Ponraj Prabakaran, Dimiter S DimitrovAbstract:Combinatory antibody library Display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a Yeast Display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a Yeast Display naïve antibody library. All the Yeast Display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins.
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Yeast Display of engineered antibody domains
Methods of Molecular Biology, 2012Co-Authors: Qi Zhao, Zhongyu Zhu, Dimiter S DimitrovAbstract:Yeast Display is an efficient technology for selection of antibodies and other proteins with high affinity and thermal stability. Here, we describe a method for affinity maturation of engineered antibody domains (eAds) using Yeast Display. EAd Yeast libraries of relatively large size (∼10⁹) were generated and subjected to alternating rounds of magnetic-activated cell sorting (MACS), fluorescent-activated cell sorting (FACS), and random mutagenesis. The highest affinity clones from the final round of maturation were identified and analyzed. We discuss extensively each key step, and provide detailed protocols and helpful notes.
Dane K Wittrup - One of the best experts on this subject based on the ideXlab platform.
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a switchable Yeast Display secretion system
Protein Engineering Design & Selection, 2015Co-Authors: James A Van Deventer, Ryan L Kelly, Saravanan Rajan, Dane K Wittrup, Sachdev S SidhuAbstract:Display technologies such as Yeast and phage Display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of Displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a Yeast-based switchable Display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between Display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable Display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation.
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high affinity t cell receptors from Yeast Display libraries block t cell activation by superantigens
Journal of Molecular Biology, 2001Co-Authors: Michele C Kieke, Eric V Shusta, Dane K Wittrup, Eric J Sundberg, Roy A Mariuzza, David M KranzAbstract:The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of Yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.
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in vitro evolution of a t cell receptor with high affinity for peptide mhc
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Phillip D Holler, Eric V Shusta, David M Kranz, Dane K Wittrup, Philmore O Holman, Sean OherrinAbstract:T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using Yeast Display and selection from a library of Vα CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (KD ≈ 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3α that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.
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25 Yeast surface Display for directed evolution of protein expression affinity and stability
Methods in Enzymology, 2000Co-Authors: Eric T. Boder, Dane K WittrupAbstract:Summary The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate Yeast Display mutants is typically 2–3 weeks. Each of the experimental approaches required for this method is fairly standard: Yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast Display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to Yeast two-hybrid or phage Display methodologies.
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Yeast polypeptide fusion surface Display levels predict thermal stability and soluble secretion efficiency
Journal of Molecular Biology, 1999Co-Authors: Eric V Shusta, David M Kranz, Michele C Kieke, Evan A Parke, Dane K WittrupAbstract:Efficiency of Yeast cell surface Display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable Yeast surface Display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the Yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in Yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR Displayed as a fusion to Aga2p on the surface of Yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates Display efficiency. Thus, Yeast Display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.
