Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries.

Cyclic peptides with engineered protein-binding activity have great potential as therapeutic and diagnostic reagents owing to their favorable properties, including high affinity and selectivity. Cyclic peptide binders have generally been isolated from phage display combinatorial libraries utilizing panning based selections. As an alternative, we have developed a yeast surface display platform to identify and characterize cyclic peptide binders from genetically encoded combinatorial libraries. Through a combination of magnetic selection and fluorescence-activated cell sorting (FACS), high-affinity cyclic peptide binders can be efficiently isolated from yeast display libraries. In this platform, linear peptide precursors are expressed as yeast surface fusions. To achieve cyclization of the linear precursors, the cells are incubated with disuccinimidyl glutarate, which crosslinks amine groups within the displayed linear peptide sequence. Here, we detail protocols for cyclizing linear peptides expressed as yeast surface fusions. We also discuss how to synthesize a yeast display library of linear peptide precursors. Subsequently, we provide suggestions on how to utilize magnetic selections and FACS to isolate cyclic peptide binders for target proteins of interest from a peptide combinatorial library. Lastly, we detail how yeast surface displayed cyclic peptides can be used to obtain efficient estimates of binding affinity, eliminating the need for chemically synthesized peptides when performing mutant characterization.

Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.

The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of target-displaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry.

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 µM for YAP and 0.68 µM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics.

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as ß-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.

Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands.

This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of select WW-YAP-binding peptides: KD values of 37 and 4 µM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 µM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ∼ 1.2-3.7 µM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity (KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

Past, Present, and Future of Affinity-based Cell Separation Technologies.

Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.

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.

Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.

An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells.

Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.

Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics.

With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such as surface plasmon resonance.

Single-Particle Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors.

Determining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons-a newly recognized CPV host-to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity.