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A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli -based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the DVPDY sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies. [ABSTRACT FROM AUTHOR]

The pentaspan transmembrane glycoprotein CD133, prominin-1, is expressed in cancer stem cells in many tumors and is promising as a novel target for the delivery of cytotoxic drugs to cancer-initiating cells. In this study, we prepared a mouse library of single-chain variable fragment (scFv) antibodies using mRNAs isolated from mice immunized with the third extracellular domain of a recombinant CD133 (D-EC3). First, the scFvs were directly exposed to D-EC3 to select a new specific scFv with high affinity against CD133 using the ribosome display method. Then, the selected scFv was characterized by the indirect enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and in silico analyses included molecular docking and molecular dynamics simulations. Based on ELISA results, scFv 2 had a higher affinity for recombinant CD133, and it was considered for further analysis. Next, the immunocytochemistry and flow cytometry experiments confirmed that the obtained scFv could bind to the CD133 expressing HT-29 cells. Furthermore, the results of in silico analysis verified the ability of the scFv 2 antibody to bind and detect the D-EC3 antigen through key residues employed in antigen–antibody interactions. Our results suggest that ribosome display could be applied as a rapid and valid method for isolation of scFv with high affinity and specificity. Also, studying the mechanism of interaction between CD133's scFv and D-EC3 with two approaches of experimental and in silico analysis has potential importance for the design and development of antibody with improved properties. [ABSTRACT FROM AUTHOR]

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research. [ABSTRACT FROM AUTHOR]

Abstract Molecular imaging plays important roles in many fields, including disease diagnosis, therapeutic efficacy evaluation, intraoperative imaging guidance, drug metabolism monitoring, and patient selection for appropriate treatment. As a key component, the targeting ligand determines the specificity, affinity, and in vivo performance of molecular imaging probes. In this review, high‐throughput screening and biological display platforms for the discovery of ligands applicable to molecular imaging are briefly reviewed. Basic information on ligand development for molecular imaging is first introduced, followed by a presentation of various selection platforms and typical or iterative cases. The features, advantages, limitations, and application scope of screening and display platforms are compared and discussed. Last, a basic selection strategy and a perspective for protein‐based ligands are provided.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors. [ABSTRACT FROM AUTHOR]

We combined cell‐free ribosome display and cell‐based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL‐9Rα). The target, IL‐9Rα, is the receptor involved in the signalling pathway of IL‐9, a pro‐inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths—the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target. [ABSTRACT FROM AUTHOR]

Currently, various pharmaceutical modalities are being developed rapidly. Targeting protein–protein interactions (PPIs) is an important objective in such development. Cyclic peptides, because they have good specificity and activity, have been attracting much attention as an alternative to antibody drugs. However, cyclic peptides involve some difficulties, such as oral availability and cell permeability. Therefore, while small-molecule drugs still present many benefits, the screening of functional small-molecule compounds targeting PPIs requires a great deal of time and effort, including structural analysis of targets and hits. In this study, we investigated a rational two-step strategy to design small-molecule compounds targeting PPIs. First, we obtained inhibitory cyclic peptides that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) by ribosomal display using PUREfrex® (PUREfrex®RD) to get structure–activity relation (SAR) information. Based on that information, we converted cyclic peptides to small molecules using PepMetics® scaffolds that can mimic the α-helix or β-turn of the peptide. Finally, we succeeded in generating small-molecule compounds with good IC50 (single-digit μM values) against CTLA-4. This strategy is expected to be a useful approach for small-molecule design targeting PPIs, even without having structural information such as that associated with X-ray crystal structures. [ABSTRACT FROM AUTHOR]

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest.

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources. [ABSTRACT FROM AUTHOR]

Sea lamprey immunization can yield leucine-rich repeat (LRR) protein binders analogous to globular antibodies developed from mammals. A novel minimal LRR was discovered through lamprey immunization with human immunoglobulin G Fc domain (IgG Fc). Initial attempts to solubly express this LRR protein, VLRB.IgGFc, in Escherichia coli proved challenging, so it was analyzed using the cell-free method ribosome display. In ribosome display, VLRB.IgGFc was found to bind specifically to the Fc domain of IgG, with little observed cross-reactivity to IgA or IgM. The minimal repeat protein architecture of VLRB.IgGFc may facilitate modular LRR extensions to incorporate additional or augmented functionality within a continuous, structurally defined scaffold. We exploited this modularity to design a chimera of a well-characterized, soluble LRR repebody and the initially insoluble VLRB.IgGFc to produce soluble Repe-VLRB.IgGFc. The minimal IgG Fc–binding module, Repe-VLRB.IgGFc, and future-engineered variants thereof should be useful additions to the biotechnological toolbox for detecting, purifying, or targeting IgGs. More generally, this two-step approach of minimal LRR binder discovery via sea lamprey immunization followed by modular augmentation of functionality may be of general utility in protein engineering. [ABSTRACT FROM AUTHOR]

In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody‐like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allow in vitro transcription and translation using an Escherichia coli lysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino‐functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA–ribosome–antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin‐binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50 values as low as 14.039 ± 0.970 ng/mL were identified. Moreover, the limits of detection reached 0.083 ± 0.011 ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance. [ABSTRACT FROM AUTHOR]

Glucose-regulated protein 78 (GRP78) is a well-characterized endoplasmic reticulum (ER) chaperon frequently overexpressed at the surface of tumor cells and associated with tumor survival, metastasis, and chemoresistance. Hence, potential GRP78 binders emerge as promising candidates for cancer therapy and diagnosis. We applied ribosome display to isolate a single-chain variable domain (scFv) specific for the C-terminal domain of a recombinant human GRP78 (CGRP). Six female BALB/c mice were immunized and then splenocyte mRNA was extracted. An scFv-ribosome display library was established by joining the amplified VH/Vκ fragments through a 72-bp linker using overlap extension PCR. Then, selection was performed by applying two rounds of eukaryotic ribosome display panning with stepwise decreased amount of CGRP. Ultimately, the selected scFv was characterized using the indirect-ELISA assay, competitive-ELISA assay, Western blotting, Surface Plasmon Resonance (SPR), and in-silico analyses. The constructed library had a length of ~ 1100 bp and the high-affinity scFvs were isolated using the outputs of the final panning round. Among 60 positive clones, GSF3 was selected and its expression, purification, and binding capacity was confirmed by SDS-PAGE and Western blotting. GSF3 exhibited an affinity of 13 × 107 M-1 to CGRP as assessed by SPR. Moreover, the in-silico analyses indicated that GSF3 binds the C-terminal domain of GRP78 through key residues engaged in antibody-antigen interactions. We found that ribosome display is a swift and reliable technique for specific and high-affinity scFv isolation. Moreover, our results suggest that GSF3 might be applied as a potential cancer immunotherapeutic and diagnostic tool if this approach is carefully followed by successful preclinical and clinical evaluations to validate the findings for further confirmation. [ABSTRACT FROM AUTHOR]

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggeredextension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity. [ABSTRACT FROM AUTHOR]

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies. [ABSTRACT FROM AUTHOR]

Objective(s): Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the 6-kDa early secretory antigenic target (ESAT-6) antigen of Mycobacterium tuberculosis from a mouse scFv library. Materials and Methods: The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in Escherichia coli BL21 (DE3). The purified recombinant ESAT-6 protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 using a single round of ribosome display via a rabbit reticulocyte lysate system. Results: ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-6 protein. The affinity of the candidate scFv was ̴ 3.74×108 M-1. Conclusion: It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.

Monoclonal antibody biologics have significantly transformed the therapeutic landscape within the biopharmaceutical industry, partly due to the utilisation of discovery technologies such as the hybridoma method and phage display. While these established platforms have streamlined the development process to date, their reliance on cell transformation for antibody identification faces limitations related to library diversification and the constraints of host cell physiology. Cell-free systems like ribosome display offer a complementary approach, enabling antibody selection in a completely in vitro setting while harnessing enriched cellular molecular machinery. This review aims to provide an overview of the fundamental principles underlying the ribosome display method and its potential for advancing antibody discovery and development.

Endemic countries with lymphatic filariasis are striving towards the Global Program to Eliminate Lymphatic Filariasis (GPELF) by 2020. Efficient and cost-effective diagnostic tools to assess active filarial infection are critical to eradicate lymphatic filariasis. Detection of circulating filarial antigens in sera is one of the precise methods to identify this infection. Monoclonal antibodies and single chain fragment variable (scFv) against Wuchereria bancrofti antigen SXP1 have been developed for antigen detection. Molecular cloning of scFv for recombinant expression has laid a platform for developing novel genetic constructs with enhanced reactivity. In this study, a simple procedure is developed to create diverse libraries of scFv based on a single DNA framework with all the requisites for an in vitro protein synthesis and ribosomal display. Error Prone-PCR was performed to incorporate random mutations and screened by ribosome display technique to isolate evolved scFv. Evolved scFv with six mutations showed tenfold increase in affinity compared to wild-type scFv for rWbSXP1. In silico studies showed that four mutations introduced unique molecular interactions between the evolved scFv and SXP1. Reactivity with asserted clinical samples of endemic normals (EN), microfilariaemic (MF), chronic pathology (CP) and non-endemic normals (NEN) showed significant augment (59.69%, p < 0.0001) in reactivity to MF samples with evolved scFv in comparison to wild-type scFv. Sensitivity of scFv was increased from 15.62 ng to 195 pg by evolved scFv in serum samples. This evolutionary method coupled with ribosome display has facilitated us to improve the reactivity of the ScFv without diminishing the specificity. [ABSTRACT FROM AUTHOR]

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole‐living‐cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (Tm = 77.0°C). Anti‐S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead‐fishing, and fluorescence imaging. Thus, novel anti‐bacteria Affitins which are cost‐effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface‐exposed marker. This class of reagents can be useful in diagnostic and biomedical applications. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Coupling ribosome display to NGS allowed identifying specific anti-CTX-M15 affibodies. • Affibody ligand is able to detect CTX-M15 in simple immune-based tests. • Affibody binding leads to a decrease of CTX-M15 substrate affinity. Abstract CTX-M15 is one of the most widespread, extended spectrum β-lactamases, a major determinant of antibiotic resistance representing urgent public health threats, among enterobacterial strains infecting humans and animals. Here we describe the selection of binders to CTX-M15 from a combinatorial affibody library displayed on ribosomes. Upon three increasingly selective ribosome display iterations, selected variants were identified by next generation sequencing (NGS). Nine affibody variants with high relative abundance bearing QRP and QLH amino acid motifs at residues 9–11 were produced and characterized in terms of stability, affinity and specificity. All affibodies were correctly folded, with affinities ranging from 0.04 to 2 μM towards CTX-M15, and successfully recognized CTX-M15 in bacterial lysates, culture supernatants and on whole bacteria. It was further demonstrated that the binding of affibody molecules to CTX-M15 modulated the enzyme's kinetic parameters. This work provides an approach using ribosome display coupled to NGS for the rapid generation of protein ligands of interest in diagnostic and research applications. [ABSTRACT FROM AUTHOR]

Ribosome display is a powerful engineering research tool for the high-throughput selection of peptides or proteins, which results in the generation of high-performance binders against nearly any antigen of interest. As a cell-free display system, ribosome display has been well developed with many outstanding achievements for over 20 years. Compared with other related display techniques, ribosome display shows unique advantages and development prospects. This tool has been successfully exploited for the selection of functional and specific binders in vitro. This review provides a comprehensive survey of the applications of ribosome display in screening or evolving functional proteins as well as in diagnostics and therapeutics. Previous papers on ribosome display failed to comprehensively review evolutionary strategies for proteins. In the present paper, we review all existing evolutionary strategies that have been combined with ribosome display. We also discuss shortcomings, improvement strategies, and research tendency. [ABSTRACT FROM AUTHOR]

HER2 is a member of epidermal factor receptor (EGFR) family which is overexpressed in breast cancer, ovarian cancer and gastric cancer. Development of new binders for cancer cell surface receptors and expressing them at the surface of exosomes would be a great approach in targeted cancer therapy. We found a high affinity scFv against HER2 using ribosome display with the approach of applying it as a targeting moiety at the surface of exosomes by fusion to lysosomal associated membrane protein 2B (LAMP2B). We also provide some structural information about the ribosome display selected scFv (scFv HFS2) through modeling the 3D structure of scFv HFS2 using RosettaAntibody and docked it at the extracellular domain of HER2. We also evaluated the structure of scFv HFS2 and its binding to HER2 after fusion to LAMP2B. Our results showed no significant change in 3D structure of scFv HFS2 when fused to LAMP2B (RMSD 1.3) and interaction analysis represented that scFv HFS2 binds HER2 domain III before and after fusion to LAMP2B. Although binding domain of scFv HFS2 on HER2 was the same at both state, residues involved in their interactions showed significant differences as it was probably due to the spatial hindrance of scFv HFS2 when fused to LAMP2B through a short linker and it should be considered before proceeding to experiment. [ABSTRACT FROM AUTHOR]

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

[Display omitted] • Major/Minor Translocator-chaperone complexes are crucial for effective T3SS operation. • Specificity of P. aeruginosa major/minor chaperone-translocator complexes investigated. • Ribosome displayed chaperone-based libraries were panned against both translocators. • Each translocator select tight binding motifs at N-terminal/convex surface interfaces. • Results identify key WT interactions & novel non-WT chaperone sequences. One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial "toxins" via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator–chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH's N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates. [ABSTRACT FROM AUTHOR]

Antibody ribosome display remains one of the most successful in vitro selection technologies for antibodies fifteen years after it was developed. The unique possibility of direct generation of whole proteins, particularly single-chain antibody fragments (scFvs), has facilitated the establishment of this technology as one of the foremost antibody production methods. Ribosome display has become a vital tool for efficient and low-cost production of antibodies for diagnostics due to its advantageous ability to screen large libraries and generate binders of high affinity. The remarkable flexibility of this method enables its applicability to various platforms. This review focuses on the applications of ribosome display technology in biomedical and agricultural fields in the generation of recombinant scFvs for disease diagnostics and control.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Distinguishing the surface markers of cancer stem cells (CSCs) is a useful method for early diagnosis and treatment of tumors, as CSCs may participate in tumorigenesis and metastasis by migrating into the circulatory system. However, the potential targets of CSCs are expressed at low levels in the natural state and are always changing. Thus, dynamic screening has been reported to be an effective measure for exploring CSC markers. In recent years, diverse single-chain variable fragments (scFvs) have been widely used in immunotherapy. In this study, we determined that the scFvs, screened using RD, had a high affinity to microspheres and could inhibit their progression. We also observed that the selected scFvs underwent evolution in vitro, and antitumor-associated proteins were successfully expressed. Combined with chemotherapy, the scFvs had a synergistic effect on the inhibition of the microspheres' progression in vitro and in vivo, which could be ascribed to their high affinity for stem-like cells and the inhibition of the microspheres' collective behaviors. In addition, proteins inhibiting CD44+/CD24+ and MAPK were involved. Our data indicated that dynamic screening of the scFvs in a natural state was of great significance in the inhibition of the microspheres in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities ( KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids). [ABSTRACT FROM AUTHOR]

The two most used methods to select camelid single-domain antibody-fragments (VHHs) are: displaying their repertoires on the surface of filamentous bacteriophages (phage display) or linking them to ribosomes (ribosome display). In this study, we compared specific VHHs isolated from two different immune libraries coming from two different alpacas by using these two selection methods. Three anti-GFAP (glial fibrillary acidic protein) VHHs were derived from an immune library obtained by ribosome display after immunization of one alpaca with purified GFAP, a protein expressed by astroglial cells. In parallel, three other anti-GFAP VHHs were derived from an immune library by phage display after immunization of another alpaca with a human brain tissue extract containing GFAP. All the VHHs were closely related and one VHH was found to be strictly identical in both studies. This highlights the selection pressure exerted by the camelid immune system to shape the paratope of an antibody against a defined antigen. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) Tat is a virus-encoded trans-activator that plays a central role in viral transcription. We used our recently developed parallel analysis of in vitro translated open reading frames (ORFs) (PLATO) approach to identify host proteins that associate with HIV-1 Tat. From this proteomic assay, we identify 89 Tat-associated proteins (TAPs). We combine our results with other datasets of Tat or long terminal repeat (LTR)-associated proteins. For some of these proteins (NAT10, TINP1, XRCC5, SIN3A), we confirm their strong association with Tat. These TAPs also suppress Tat-mediated HIV-1 transcription. Removing suppression of HIV-1 transcription benefits the reversal of post-integrated, latent HIV-1 proviruses. We demonstrate that these transcriptionally suppressing TAPs contribute to HIV-1 latency in Jurkat latency (J-LAT) cells. Therefore, our proteomic analysis highlights the previously unappreciated TAPs that play a role in maintaining HIV-1 latency and can be further studied as potential pharmacological targets for the "shock and kill" HIV-1 cure strategy. [ABSTRACT FROM AUTHOR]

Objective(s): Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the 6-kDa early secretory antigenic target (ESAT-6) antigen of Mycobacterium tuberculosis from a mouse scFv library. Materials and Methods: The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in Escherichia coli BL21 (DE3). The purified recombinant ESAT-6 protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 using a single round of ribosome display via a rabbit reticulocyte lysate system. Results: ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-6 protein. The affinity of the candidate scFv was~ 3.74×108 M-1. Conclusion: It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB. [ABSTRACT FROM AUTHOR]

Protein-protein interactions are fundamental for virtually all functions of the cell. A large fraction of these interactions involve short peptide motifs, and there has been increased interest in targeting them using peptide-based therapeutics. Peptides benefit from being specific, relatively safe, and easy to produce. They are also easy to modify using chemical synthesis and molecular biology techniques. However, significant challenges remain regarding the use of peptides as therapeutic agents. Identification of peptide motifs is difficult, and peptides typically display low cell permeability and sensitivity to enzymatic degradation. In this review, we outline the principal high-throughput methodologies for motif discovery and describe current methods for overcoming pharmacokinetic and bioavailability limitations. [ABSTRACT FROM AUTHOR]