Application of the yeast‐surface‐display system for orally administered salmon calcitonin and safety assessment

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface‐displayed Saccharomyces cerevisiae as a low‐cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M‐pYD1 to yield recombinant yAGA2‐sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2‐sCT to Wistar rats, respectively. For safety assessment of yAGA2‐sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2‐sCT induced a long‐term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2‐sCT anchoring sCT protein on a S. cerevisiae surface has potential for low‐cost and safe oral delivery of sCT. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

Constraints imposed by non‐functional protein–protein interactions on gene expression and proteome size

Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing non‐functional interactions with promiscuous non‐functional partners. Here we show how the need to minimize the waste of resources to non‐functional interactions limits the proteome diversity and the average concentration of co‐expressed and co‐localized proteins. Using the results of high‐throughput Yeast 2‐Hybrid experiments, we estimate the characteristic strength of non‐functional protein–protein interactions. By combining these data with the strengths of specific interactions, we assess the fraction of time proteins spend tied up in non‐functional interactions as a function of their overall concentration. This allows us to sketch the phase diagram for baker's yeast cells using the experimentally measured concentrations and subcellular localization of their proteins. The positions of yeast compartments on the phase diagram are consistent with our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, whereas keeping individual protein concentrations sufficiently low to reduce non‐functional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity and differentiation.     

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha‐helical ubiquitin interacting motif

Ubiquitin interacting motifs (UIMs) are short α‐helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N‐terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1‐UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α‐helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.     

Yeast display platform with expression of linear peptide epitopes for high-throughput assessment of peptide-MHC-II binding

Yeast display can serve as a powerful tool to assess the binding of peptides to the major histocompatibility complex (pMHC) and pMHC-T-cell receptor binding. However, this approach is often limited by the need to optimize MHC proteins for yeast surface expression, which can be laborious and may not yield productive results. Here we present a second-generation yeast display platform for class II MHC molecules (MHC-II), which decouples MHC-II expression from yeast-expressed peptides, referred to as “peptide display.” Peptide display obviates the need for yeast-specific MHC optimizations and increases the scale of MHC-II alleles available for use in yeast display screens. Because MHC identity is separated from the peptide library, a further benefit of this platform is the ability to assess a single library of peptides against any MHC-II. We demonstrate the utility of the peptide display platform across MHC-II proteins, screening HLA-DR, HLA-DP, and HLA-DQ alleles. We further explore parameters of selections, including reagent dependencies, MHC avidity, and use of competitor peptides. In summary, this approach presents an advance in the throughput and accessibility of screening peptide-MHC-II binding.     

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.     

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Preferential selection of Cys‐constrained peptides from a random phage‐displayed library by anti‐glucitollysine antibodies

Phage‐displayed peptides recognized by two monoclonal antibodies against glucitollysine were selected. The most prominent feature of the peptide panel was the presence of paired Cys in most of them (21/24 peptides). The availability of a wide variety of peptides having differently spaced paired Cys, as well as truly linear Cys‐free peptides, gave the opportunity to explore the role of disulfide bridges in phage selection. Some Cys‐containing peptides came from a Cys‐flanked cyclic 9‐mer library, but most of them (18/21) were derived from a totally random 12‐mer library, and hence the presence of Cys was dictated by the selector antibodies. Motifs shared by several peptides (potentially involved in binding) often contained or were flanked by Cys residues. Binding of all Cys‐containing phage‐displayed peptides was abolished/decreased after a reducing treatment. Screening a random peptide library (without invariant Cys residues) is powerful enough to clearly reveal the need, preferences, and diversity of Cys‐mediated structural constraints for recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (k_off) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20–40% improvement in the measured k_off. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t_½) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

An engineered tryptophan zipper‐type peptide as a molecular recognition scaffold

In an effort to develop a structured peptide scaffold that lacks a disulfide bond and is thus suitable for molecular recognition applications in the reducing environment of the cytosol, we investigated engineered versions of the trpzip class of β‐hairpin peptides. We have previously shown that even most highly folded members of the trpzip class (i.e. the 16mer peptide HP5W4 ) are substantially destabilized by the introduction of mutations in the turn region and therefore not an ideal peptide scaffold. To address this issue, we used a FRET‐based live cell screening system to identify extended trpzip‐type peptides with additional stabilizing interactions. One of the most promising of these extended trpzip‐type variants is the 24mer xxtz1 ‐peptide with the sequence KAWTHDWTWNPATGKWTWLWRKNK. A phage display library of this peptide with randomization of six residues with side chains directed towards one face of the hairpin was constructed and panned against immobilized streptavidin. We have also explored the use of xxtz1 ‐peptide for the presentation of an unstructured peptide ‘loop’ inserted into the turn region. Although NMR analysis provided no direct evidence for structure in the xxtz1 ‐peptide with the loop insertion, we did attempt to use this construct as a scaffold for phage display of randomized peptide libraries. Panning of the resulting libraries against streptavidin resulted in the identification of peptide sequences with submicromolar affinities. Interestingly, substitution of key residues in the hairpin‐derived portion of the peptide resulted in a 400‐fold decrease in K_d, suggesting that the hairpin‐derived portion plays an important role in preorganization of the loop region for molecular recognition. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation of Common Light Chain Antibodies from Immunized Chickens Using Yeast Biopanning and Fluorescence-Activated Cell Sorting

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)-based chicken monoclonal antibodies from an anti-epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence-activated cell sorting (FACS). For the selection of high-affinity antibodies, a yeast cell library presenting cLC-comprising fragment antigen binding (Fab) fragments is panned against hEGFR-overexpressing A431 cells. The resulting cell–cell-complexes are sorted by FACS resulting in gradual enrichment of EGFR-binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC-based bispecific antibodies against native mammalian receptors.     

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins.     

Consequences of stress induced by heat on fitness of Delphacodes kuscheli and its yeast‐like endosymbionts

The planthopper Delphacodes kuscheli is the main vector of Mal de Río Cuarto virus in Argentina, disease that severely affects maize production. In this study, we investigated the effects of heat stress on fitness traits and on the number of its obligate yeast‐like symbionts (YLS). The exposition of newly‐hatched nymphs to 35°C for 3 days, a well‐known procedure used to reduce the number of YLS in planthoppers, was applied. To compare different fitness components between control and heat‐treated insects, we estimated nymphal instars development time, nymphal survival, adult body length, longevity, fecundity and fertility. Also, correlates of fitness, as proportion of sexes and wing forms of the emerging adults, were evaluated. In heat‐treated group, the nymphal developmental time increased due to an increase in the fifth instar duration, and the nymphal survival, body length of adults and fecundity were reduced when compared to control. There was a significant association between treatments (control and heat‐treated insects) and wing morphs. The heat treatment successfully reduced the number of YLS in third instar nymphs of D. kuscheli. Our results revealed the negative effect of heat stress on development, survival and reproduction of D. kuscheli and on the load of its YLS endosymbionts suggesting that YLS could play a crucial role in the development and reproduction of these planthoppers.     

A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high‐affinity Aβ‐binding molecules were isolated using flow‐cytometric sorting. The best performing candidate binds Aβ with a K_D value of around 300 pM, corresponding to a 50‐fold improvement in affinity relative to the first‐generation binder. The new dimeric Affibody molecule was shown to capture Aβ_1‐42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.     

Establishment of cell surface engineering and its development

Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.     

Over‐expression of secreted proteins from mammalian cell lines

Secreted mammalian proteins require the development of robust protein over‐expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large‐scale growth of cells in a variety of formats.