phage display system for protein engineering

Nagano K., Tsutsumi Y. Phage display technology as a powerful platform for antibody drug discovery. In the native form, many such linear epitopes are often hidden or interrupted, and outcompeted by the three-dimensional epitopes exposed and more potent for in vitro selection. Further development of this approach for more biological systems could in principle advance analysis of the energy landscapes of many systems including highly challenging regulatory proteins that control physiological responses to environmental changes. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Krishnaswamy S., Kabir M. E., Miyamoto M., Furuichi Y., Komiyama T. Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution. Bethesda, MD 20894, Web Policies Phage display: protein engineering by directed evolution However, despite of all the advantageous features of other display techniques mentioned above, phage display remains the most popular display platform today. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Bashir S., Paeshuyse J. Yu Y., Zheng Q., Erramilli S. K., Pan M., Park S., et al. Engin S., Fichtner D., Wedlich D., Fruk L. SNAP-tag as a tool for surface immobilization. 5) Amplification of the enriched phage by propagation in Escherichia Although, due to the new algorithms and advances in computer performance, novel and improved synthetic protein structures and functions can now be designed entirely in silico by rational molecular and de novo design [2], synergistic combination of the computational design and in vitro evolutionary approaches produces variants superior to those that could be generated by the design only [3]. The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab. Molecular displays on the surface of eukaryotic cells are highly permissive for multi-domain proteins and multi-molecular complexes, as well as for synthesis of functional mammalian proteins undergoing essential posttranslational modifications that are supported by the eukaryotic cytoplasm [45]. Hundreds of the single-stranded copies of the M13 genome are made by the mechanism called rolling-circle replication using RF DNA as a template: some of them are converted into more RF molecules, while the majority are destined to be packaged into the phage progeny. HHS Vulnerability Disclosure, Help For the purpose of application in phage display, the scaffold proteins of the nanodiscs are biotinylated enabling their pull-out by the magnetic beads. This gp5 shell packs genomic DNA into a flexible rod protected from nucleases, thus forming the intracellular precursor of the extracellular virion. 3). 1a) Notably, while Fabs generally maintain full antigen-binding affinity upon reformatting into the full-size IgG and back (taking into account the 2-fold difference in the format valency), the scFv affinity can be drastically changed upon reformatting and sometimes is influenced by the VH/VL domain mutual orientation and/or linker length, and, also, this format is prone to oligomerization [34, 35]. Phage display technology can produce a. The alternative cell-free methods, like ribosome display [46] and mRNA display [47] offer their own advantages, such as speedy turnover, enormous library size, and potential incorporation of non-natural amino acids and chemical modifications into the proteins. Aiming for the library size 1010 unique CDR combinations can significantly improve the clone coverage and sampling power of the phage display. Rizk S. S., Kouadio J. L., Szymborska A., Duguid E. M., Mukherjee S., et al. Structure determination of small biological complexes using single-particle cryo-EM. Hormone phage: An enrichment method for variant proteins with altered binding properties. Protein posttranslational modifications: the chemistry of proteome diversifications. Selective enrichment and high-throughput screening of phage surface-displayed cDNA libraries from complex allergenic systems. An engineered GA1 variant. Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy. These would eliminate the need to incorporate the challenge of an antibody-engineering into the workflow of protein-structure determination projects. A naturally occurring analog to phage display, the Bordetella bronchiseptica bacteriophage (BP) employs a highly variable protein termed the major tropism determinant (Mtd) to recognize its dynamic host. Given the ease of interchanging FabLRT in the bi-Fabs, we envision the following possible advances of the GA1-based plug-and-play BiTE mimetics. Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin. Yeast Surface Display System for Facilitated Production and - PubMed Another, the least structure-disturbing way for biotin attachment, is through a genetic linkage of the antigen to Avi-tag, a peptide (GLNDIFEAQKIEWHE) specifically recognized and efficiently biotinylated by the biotin ligase BirA. There is a great variety of possible functionally relevant GA1 genetic fusions, allowing the researcher to design highly customized affinity reagents that can be assembled in a wide scope of different formats in a straightforward way (Fig. Due to the same AB scaffold used for cloning of all the variants, sABs are easy to sequence and reformat into a full antibody or expression vectors for production in bacterial or mammalian cells. The resulting immune library does not need to be as large as nave libraries, since the immunized pool of lymphocytes would contain multiple proliferated B-cell clones targeting the antigen. This direct covalent capture could be hugely beneficial for the proteins with IMAC-incompatible characteristics or complicated purification protocols, despite the fact, that, in our hands, the SNAP-capture beads demonstrated reproducibly somewhat lower antigen-binding capacity and specificity for phage binding, compared to the best-performing Streptavidin-coupled magnetic beads (Dyno beads, Invitrogen, USA). Construction of the phage-display library for Fab scaffold GA1-binding affinity maturation. The detection limit of the systems (~10 nM) falls within the standard range for a sandwich antigen-detection immunoassay, like ELISA. Recognition of an alpha-helical hairpin in P22 large terminase by a synthetic antibody fragment. The resulting novel, easily manipulatable, multifunctional, and sturdy GA1-based plug-and-play platform utilizing FabLRT molecules as the interchangeable elements, and its possible applications in basic science, biotechnology, and medicine are considered in the conclusion. Rapid discovery and characterization of synthetic neutralizing antibodies against Anthrax Edema toxin. Phage Display Systems for Protein Engineering - Creative Biolabs Sophisticated interrelation between the structural and functional transformations underlying the transduction process is one of the most complicated though most exciting areas of the todays membrane-protein science. Nuclear pores. To be displayed, usually the antibody fragment is fused to the N-termini of gp3 or its C-terminal domain, which itself assembles into a capsid tip, while it is the N-terminal domain of gp3, which recognizes the E. coli F-pilus. 4d) has been developed by fusing GA1 to two split -lactamase complementation fragments [158] (details of this work are discussed in the next chapter). Morag O., Sgourakis N. G., Baker D., Goldbourt A. sABs as tools for protein structure determination. This significantly enhanced sAB specificity and solubility, crucial for easily aggregating antibodies, while maintained its affinity to the antigen [149]. Biotinylation of the SNAP-tagged protein is a bi-molecular reaction, simple, fast, completed within a short time, and irreversible that does not require any substantial excess of the SNAP-biotin reagent and, subsequently, is free of the purification step prior to the binding to streptavidin magnetic beads. A cross-species display system that allows one to exploit the strengths of prokaryotic and eukaryotic display systems would represent a significant advancement for antibody discovery and engineering. This wash-free sandwich antigen-detection system has been also applied for SARS-CoV-2 detection using a pair of FabLRT targeting two non-overlapping epitopes of the Spike RBD. Farcasanu M., Wang A. G., Uchaski T., Bailey L. J., Yue J., et al. A rapid and efficient screening to tailor phage-display for the selection of neutralizing antibody was set up in the KossLab for the Anthrax model. Nissim A., Hoogenboom H. R., Tomlinson I. M., Flynn G., Midgley C., et al. Although the FabLRTGA1 interaction is not covalent, no exchange between the partners, which could compromise the performance of such a platform, has been detected within the timeframe of experiments. When implemented in the phage display libraries, they yielded high-quality non-Ig binders that could be of great interest for basic and applied scientific research due to their robust folding, high solubility, and small size. Urh M., Rosenberg M. HaloTag, a platform technology for protein analysis. An official website of the United States government. 2016;22(43):6538-6559. doi: 10.2174/1381612822666160923113714. In the library, limited amino acid diversity is introduced into all three HC CDRs (H1, H2, and H3), as well as the third CDR of LC: L3, while L1 and L2 have fixed canonical loops: SVSSA and SASSLYS, respectively. The library design was focusing on the residues 123-127 (SQLKS) of CL. Among the set of generated actin-filament pointed-end binders, three sABs have demonstrated unique properties toward the actin-dynamic probing: one binder caps the pointed end, the second one crosslinks actin filaments, and the third severs actin filaments and promotes disassembly. Although there are many other types and varieties of protein-binding chemistry, the exceptionally-high femtomolar affinity of a biotinstreptavidin pair makes this technique generally superior to others, since dissociative loss of the target during multiple washing steps at lower immobilization affinities could ultimately impede quality of the selection. Grard A., Woolfe A., Mottet G., Reichen M., Castrillon C., et al. Dean A. Q., Luo S., Twomey J. D., Zhang B. Weinstein JB, Bates TA, Leier HC, McBride SK, Barklis E, Tafesse FG. Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G. Sheng S., Kong F. Separation of antigens and antibodies by immunoaffinity chromatography. Schematic representation of T-cell engagement in cytolytic synapse with cancer cell expressing HER2 receptor by means of bi-Fab inter-cellular bridging: it binds HER2 receptor with FabH, genetically fused to GA1, while FabLRT, non-covalently attached to GA1, binds to CD3 co-receptor of T-cell specific receptor (TCR). Moore J. C., Arnold F. H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Samish I. BirA could modify Avi-tagged proteins in vitro, after purification [105], or in vivo [106], during expression in E. coli bearing BirA plasmid; in every case high-level of antigen biotinylation has been demonstrated (70-90%). Additionally, fusion proteins could be rejected after the synthesis at different stages of phage maturation. 2016 Elsevier Inc. All rights reserved. Shukla A. K., Manglik A., Kruse A. C., Xiao K., Reis R. I., et al. Together with the simple and fast non-wash procedure, reproducible quantitative results, and detected-antigen adjustability, easily achievable by FabLRT pair exchange, this makes the protein-complementation GA1-based assay a suitable candidate for POC applications; in addition, it was shown, that its components can be freeze-dried and fully reactivated after rehydration [85]. 1b), are successfully challenging the originals. Natural antibody libraries. The antibody-based fiducial markers have considerably aided the membrane-protein structural studies in the field of G-protein coupled receptor (GPCRs) [142-144]. SNAP-tagged targets: immobilization and specific proteolytic elution. Zhou C., Yan Y., Fang J., Cheng B., Fan J. Multiple modifications of the original technology of peptide phage display, including cyclic and artificially linked peptides [23], resulted in varieties of the cancer-specific ligands validated in cancer diagnostics and therapy [24]. Antibody-displaying phage libraries can be either natural or synthetic, or both, depending on the origin of the diversity component, incorporated into the antibody-fragment scaffold. (2018) Kinetic Analysis and Epitope Binning Using Surface Plasmon Resonance, in. Briney B., Inderbitzin A., Joyce C., Burton D. R. Commonality despite exceptional diversity in the baseline human antibody repertoire. Enzymes produced through directed evolution are used to manufacture everything from biofuels to pharmaceuticals, and antibodies evolved using phage display combat autoimmune diseases and cure cancer [1]. Kirley T. L., Greis K. D., Norman A. Also, linking of GA1 to the IgG Fc fragment replacing Fab, resulted in the IgG mimetic (Fig. Phage display has become established as a powerful protein engineering method for identifying polypeptides with novel properties, and altering the properties of existing ones. Since this phagemid mimics RF of the phage DNA, the phagemid-transformed F pilus + cells can efficiently produce ss phagemid DNA and encapsidate it into the infectious phage progeny with the assistance of the M13 helper phage infection that provides all the phage proteins required for phage biogenesis. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. sharing sensitive information, make sure youre on a federal Although the biotinstreptavidin binding is not covalent, it serves all intents and purposes in biopanning, since the binding is almost irreversible (KD 1015 M). In the KossLab, this trick has been efficiently applied in many instances, in particular, for generation of the sABs targeting non-redundant epitopes in two viral proteins, C-terminal domain of Ebola Zaire Nucleoprotein (EBOV NPCT) and Zika methyltransferase (ZIKV MT), while developing a novel protein-complementation (PC)-based wash-free immunoassay [123], relevant for point-of-care (POC) applications and described in a separate chapter below. Protein and Antibody Engineering by Phage Display - PubMed 4e) with the interchangeable FabLRT specificity that could be utilized in place of multiple IgGs in the Fc-mediated processes. Furthermore, a panel of sABs binding to different regions of paramyxovirus envelope glycoproteins and affecting different processes of the viral entry into the cell has been used to understand the steps in viral membrane fusion leading to acute respiratory infections [125]. In addition, we found, that, like with other solubility-enhancing tag proteins (MBP, NusA, thioredoxin, GST, SUMO and Fh8 tag [107, 122]), fusion to the SNAP-tag (182 amino acid-long, well-structured, highly soluble, and stable protein) can drastically improve stability, solubility, and production of the hard-to-express, poorly folded, and structurally unstable proteins. Petrenko V. A., Smith G. P. Phages from landscape libraries as substitute antibodies. Single domains from a number of almost identical entities comprising multidomain bacterial protein A (Staphylococcus aureus), protein G (Streptococcus species C and G), and protein L (Peptostreptococcus magnus) have been characterized in detail both biochemically and structurally [150]. FOIA At present, the most common immobilization method avoiding adsorption-induced conformational changes of the antigen is based on the extremely tight biotinstreptavidin interactions [102] that are widely used in various research and biotechnology applications requiring tight and specific intermolecular interactions (detection and isolation of proteins, nucleic acids and lipids, protein purification, new-generation DNA-sequencing, mass-spectrometry based proteomics and many others). See this image and copyright information in PMC. EM data confirmed efficacy of the sABs as single and dual fiducials for multiple GPCR signaling complexes [87]. Later, the tailed bacteriophages T4 and T7 have also been successfully tested as display platforms [49]. We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni.In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. Under such conditions, generation of a conformationally-selective antibody recognizing just one of the antigen states is accidental. Adv Sci (Weinh). Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. Discovery of antivirals using phage display. Would you like email updates of new search results? Over the past year, the versatility of the technology has expanded to include the development of DNA-binding proteins with novel specificities, energetics of protein folding and directed evolution of antibodies. Target immobilization and phage elution. Slezak T., Kossiakoff A. Zheng S., Sham L. T., Rubino F. A., Brock K. P., Robins W. P., et al. Antibody diversity is, mostly, present in their six complementarity determining regions (CDRs), highly variable structural loops undergoing clonal selection in the immune system. Although there is almost no risk of eluting phage incompletely, the prolonged exposures to low pH could affect phage infectibility. A novel wash-free -lactamase complementation-based detection assay (Fig. However, the M13 phage has its own pluses, making it the most developed and popular antibody-display platform to date. PDF Advances in phage display screening with technology innovation Rothe C., Urlinger S., Lhning C., Prassler J., Stark Y., et al. K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle. This has driven development and validation of the high-throughput sAB expression platforms [124]. 2011 Sep;24(9):711-9. doi: 10.1093/protein/gzr034. Baeuerle P. A., Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. In addition, as in other GA1-fusion applications, the linker length could be easily adjusted tailoring the bi-Fab for the particular antigen or even antigen epitope. Koehl A., Hu H., Maeda S., Zhang Y., Qu Q., et al.
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