phage display system for protein engineering{ keyword }

The early examples of sAB libraries incorporated random CDR sequences of different length by means of degenerate oligonucleotides synthesizes from different nucleotide mixtures at different codon positions [71]. Synthetic-antibody library construction. Single-stranded DNA with, Kunkel mutagenesis. 8600 Rockville Pike 4b) for binding multiple, same or different-specificity, FabLRT molecules for the avidity boost or randomized multi-specificity, respectively. Availability of various other SNAP substrates, besides fluorescent, like SNAP-biotin and SNAP-capture magnetic beads (NEB), allowed us to develop and successfully apply several variants of the antigen-immobilization technique based on the SNAP-tagging of target proteins. However, combinatorial arrays [37] and some other new formats, such as nanobodies representing a single VHH domain of an unusual homo-dimeric camelid IgG [38] (Fig. Most of the sAB libraries use the natural Ig framework, however, a number of modules representing protein-binding polypeptides from natural sources were engineered into the stable spatial arrangements of several diversified loops featuring compact antigen-binding structures [67]. A., Jones M. L., et al. A., Graille M., et al. 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. Covalent nature of the direct capture and extreme substrate specificity of the engineered SNAP-tag allowed for efficient immobilization of not only highly or partially purified targets but even immobilization from the crude lysates of the cells expressing SNAP-tagged proteins. official website and that any information you provide is encrypted Tools like sAB-based crystallization chaperones and fiducial markers have drastically improved the level and quality of structural analyses. Construction of naive camelids VHH repertoire in phage display-based library. Degenerate oligonucleotides are easy to design and are cost-efficient highly functional antibody libraries of 108-109 unique clones have been constructed using this method [72]. A., Rock R. S. Characterization of engineered actin binding proteins that control filament assembly and structure. We also have inserted a Thrombin-cleavage site between the tag and the antigen in the commercial SNAP-vector (New England Biolabs) to ensure fast and gentle elution of the enriched antigen-bound phage from the magnetic beads without acidic treatment. 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]. 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. 4e) with the interchangeable FabLRT specificity that could be utilized in place of multiple IgGs in the Fc-mediated processes. Vasiliauskait-Brooks I., Healey R. D., Rochaix P., Saint-Paul J., Sounier R., et al. Parmley S. F., Smith G. P. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. In this case, the sABs targeting the predominant protein epitope is pre-bound to the protein, thus, excluding the epitope from the competition for phage binding and allowing for selection of the sAB variants specific for the secondary epitopes. phage display, immunotherapy, synthetic antibody, crystallization chaperones, fiducial markers, BiTEs, bi-Fabs. Both SNAP-tag and CLIP-tag are engineered variants of the human DNA repair enzyme O(6)-alkylguanine-DNA alkylotransferase that catalyzes transfer and attachment of the alkyl group via a thioether bond to the reactive cysteine of the enzyme. Phage ELISA at single high-point concentration of phage for Dengue DIII-displaying phage (A) and protein G-displaying phage (B). Although the smaller nucleotide sets are highly desirable due to the library size limitations, there are many mutually exclusive amino acids in these sets. Are BiTEs the missing link in cancer therapy? In addition, a universal sAB-targeted tag system that eliminates the need for a specific antibody for each individual membrane protein under structural study has been developed. The 4D5 Herceptin Fab scaffold (FabS) of the KossLab pipeline containing E123S mutation in the Fab CL has been used for affinity maturation of protein G (a 65 aa-long C2 domain) [157]. 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]. Conformational chaperones for structural studies of membrane proteins using antibody phage display with nanodiscs. Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. There are other affinity tags options for tethering proteins in a relatively uniform orientation with minimal disruption of native conformation [107] including metal ion- [108, 109], antibody- [110], and proteinligand capture technologies [111] that have been used for the target immobilization. Structure determination of small biological complexes using single-particle cryo-EM. 4d) has been developed by fusing GA1 to two split -lactamase complementation fragments [158] (details of this work are discussed in the next chapter). 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. The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. The library design was focusing on the residues 123-127 (SQLKS) of CL. Kim T, Lee J, Lee JP, Kim BN, Kim YH, Lee YS, Min J. Sci Rep. 2023 Feb 6;13(1):2116. doi: 10.1038/s41598-023-28881-w. Georgiev GI, Malonis RJ, Wirchnianski AS, Wessel AW, Jung HS, Cahill SM, Nyakatura EK, Vergnolle O, Dowd KA, Cowburn D, Pierson TC, Diamond MS, Lai JR. 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. A new versatile immobilization tag based on the ultra high affinity and reversibility of the calmodulincalmodulin binding peptide interaction. Justiz Vaillant, A. A., Liu W., Roth C. B., Griffith M. T., et al. It is common, that the protein used as a selection target is not conformationally uniform and can represent a dynamic or static mix of low-energy states, sometimes caused by contaminating ligand molecules or certain metal ions. Schematic representation of the target-directed FabLRT delivering GA1-fused effector, and some effector variant (a): GA1 modules to build multimeric strings for avidity enhancement (b); enzyme- or fluorescent tags for protein detection (c), split enzymes for protein complementation detection assays (d); GA1 fusions to Fc, mimicking IgG (e); GA1 fusions with FabH (bi-Fab) or scFv for BiTE mimetics and some possible tri-specific binders (f). Protein targeting. All the bi-Fab combinations showed efficient T-cell engagement activity, similar to the activity of the covalently linked BiTE, as measured in the PBMC-SKBR3 co-cultures by three readouts: (i) activity of the cytoplasmic enzyme, lactate dehydrogenase, released into the medium upon cell lysis caused by activation of cytotoxic T-cells; (ii) interleukin IL2, and (iii) interferon produced by T-helper cells. Derebery M. J., Christopher L. Allergy, immunotherapy, alternative treatments for dizziness. Mateja A., Paduch M., Chang H. Y., Szydlowska A., Kossiakoff A. Binz H. K., Plckthun A. Surprisingly, the minimalist libraries, containing sequences of only two residues, Tyr and Ser, in their CDRs, were proven highly effective in generating specific antibodies against a wide array of antigens providing a rationale for high abundance of these two residues in the CDRs of the natural immune repertoire [62, 79]. Molecular recognition by a binary code. The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope. A mutation, that makes f1 DNA packaging signal of helper phage defective is disadvantageous for packaging of the helper-phage ssDNA, and thus favors formation of the phagemid-containing virions. Development of plug and play fiducial marks for structural studies of GPCR signaling complexes by single-particle Cryo-EM. Koldobskaya Y., Duguid E. M., Shechner D. M., Suslov N. B., Ye J., et al. 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. The most developed of those, T7 phage display, demonstrate several particular advantages over the classic M13 platform, that could be important in some special applications: (i) T7 phage contains double stranded DNA, which is more stable and less prone to mutation during replication as compared to the single-stranded M13 phage genomic DNA; (ii) foreign cDNA or bacterial genomic libraries could be directly inserted into the T7 phage ds DNA genome; (iii) T7 phage does not depend on a bacterial protein secretion pathway and has a lytic life cycle. Rigi G., Ghaedmohammadi S., Ahmadian G. A comprehensive review on staphylococcal protein A (SpA): Its production and applications. Several sABs generated and matured by the customized phage display selections against BRIL gained sub-nanomolar affinities and unhindered BRIL binding in multiple fusion systems. Antibody Phage Display: Technique and Applications - PMC In both cases, amplification of the selected molecular variants requires an unambiguous physical genotypephenotype linkage, which is readily provided by a single cell or a phage particle. determination and protein engineering. Structure of the micro-opioid receptorGi protein complex. Finally, the inner membrane inserted gp3 and gp6 proteins are attached to the proximal end of the extruding virion particle completing the phage assembly. Protein posttranslational modifications: the chemistry of proteome diversifications. Hoogenboom H. R., Griffiths A. D., Johnson K. S., Chiswell D. J., Hudson P., et al. Bai, X., and Shim, H. (2017) Construction of a scFv Library with Synthetic, Non-combinatorial CDR Diversity, in. In addition, chemical synthesis of CDRs is inherently compromised by synthetic errors, introducing all sorts of mutations that generate unwanted amino-acid substitutions and stop codons resulting in nonfunctional antibody clones [72, 77] To eliminate poorly expressed clones and non-productive frame shifts, a proofreading step, such as selection of ampicillin-resistant sAB -lactamase genetic fusions, has been added as the final step of library construction [77]. Targeted rescue of cancer-associated IDH1 mutant activity using an engineered synthetic antibody. (B) Protein G displayed as a fusion to the pIII coat protein also shows specific binding to its target IgG over negative control and displays the N-terminal FLAG-tag as probed by M2 binding. Fortunately, nature has provided protein engineers not only with the IgG molecule as a framework for sABs but also with Ig-binding proteins enabling their isolation, detection, and assembly. Phage display has been used in epitope mapping and analysis of protein-protein interactions. They can be successfully used as fiducial markers for SP cryo-EM by adding mass (50 kDa for Fab) to the particle and assisting in its orientation [129]. The KossLab pipeline production also have confirmed an undeniable potential of sABs targeting different antigen determinants in translational medicine. By its very nature, based on molecular selection by the phage binding, the phage display technology cannot address modulations and tuning of enzyme catalysis and is usually directed toward enhancement of the structural stability of the protein and binding affinities to antigens, partner proteins, or ligands, demonstrating its tremendous power pa. Phage display is a molecular biology technique by which phage genomes are modified in such a way that the coat proteins of assembled virions are fused to other proteins or peptides of interest (of any origin), displaying them thus to the external milieu. Sidhu S. S. Engineering M13 for phage display. Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. We suggest that the yeast surface display system is an efficient, stable, and straightforward platform for the production and antibacterial applications of endolysin. To further improve affinity and dissociation kinetics of the GA1Fab complex, we undertook a reciprocal phage display approach: FabS scaffold was affinity matured against GA1 [123]. Multiple GA1 modalities could be also included into the design to exploit possible avidity effects in the resulting multi-valent bi-specific formats. T select phage display system: a powerful new protein display system based on the bacteriophage T7. Also, being easily crystallizable proteins with the known scaffold structure, sABs are widely used as crystallization chaperons that bind to a target of interest, enhance crystal packing, and provide high-quality phasing information. Beyond epitope binning: Directed, Nilvebrant, J. Nonetheless, despite the constant challenge from other emerging formats, the classic libraries based on the Ig-fragment scaffolds remained the most desirable for sAb production [69]. a) Schematic structures of human IgG antibody and fragments: HC, heavy chain; LC, light chain; VH, variable domain of the heavy chain; VL, variable domain of the light chain; CH1-CH3, constant regions of the heavy chain; CL, constant domain of the light chain; Fc, fragment crystallizable; Fab, fragment antigen binding; Fd, heavy chain of the Fab; CDR, complementarity-determining regions of the heavy chain (H1-H3) and of the light chain (L1-L3); scFv, single chain fragment variable; b) camelid heavy-chain IgG antibody and VHH nanobody depictions. Epub 2021 Dec 16. Like the natural process of antibody production, in vitro selection usually generates sAB variants recognizing antigen epitopes of high immunogenicity. The N-terminal FLAG-tag can also be probed by M2 binding. Screening of novel peptides that specifically interact with vitamin D bound biocomplex proteins. George Eliava, a Georgian scientist, was one of them. 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. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling. Yeast Surface Display System for Facilitated Production and Application of Phage Endolysin ACS Synth Biol. The pooled library consists of 1010 unique variants, huge under-sampling of the theoretical diversity (by more than 20 orders of magnitude) [62]. As an in vitro method, the conditions of the binding selection can be tightly controlled. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Patel CA, Wang J, Wang X, Dong F, Zhong P, Luo PP, Wang KC. In this system, it is necessary that the protein is displayed on the . Theoretically, cleavage should release only the target-specific phage, while the SNAP-tag-bound phage should remain attached to the beads, however, natural spontaneous phage dissociation could significantly contaminate the eluted phage. The antibody-drug conjugates are able to kill cancer cells by binding to the tumor-specific receptors and directionally dispatching their payloads potent cytotoxic small molecules [25]. The matured variant GA1 demonstrated the highest improvement in the FabS binding affinity, mostly due to substitutions of NDNG in the positions 40-43 for YVHE. Phage display has become established as a powerful protein engineering method for identifying polypeptides with novel properties, and altering the properties of existing ones. (A) DIII displayed on phage binds specifically to target antibodies 4E11 and 3H5-1 but not negative control BSA. Zhou C., Yan Y., Fang J., Cheng B., Fan J. Although this system can be used to probe much larger libraries in comparison to in vivo display methods (~10 13-14 versus ~ 10 7-10 variants per . Moreover, while natural monoclonal antibody reproduction requires continuous maintenance and storage of the hybridoma cell line, sAB clones can be indefinitely preserved in a form of frozen or lyophilized DNA. 1) Target immobilization on magnetic beads. As already mentioned, sABs represent indispensable tools for protein structure determination. Protein engineering is now a mature field of protein science. Recent engineering efforts in applied immunological and biochemical research have led to production of the synthetic ligands mimicking protein A and L (peptides, engineered protein domains, and designed artificial molecules). 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. 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. However, attempts to understand molecular basis of the phenotypic alterations and to rationally design them have only become possible through the relatively recent process involving integration of the protein structure information. Nocula-Lugowska M., Lugowski M., Salgia R., Kossiakoff A. (2017) Generating Conformation and Complex-Specific Synthetic Antibodies, In: Ye J. D., Tereshko V., Frederiksen J. K., Koide A., Fellouse F. A., et al. Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2. the contents by NLM or the National Institutes of Health. Phage display: protein engineering by directed evolution Besides the checkpoint blockade, similarly to the natural monoclonal antibodies, sABs could impact a variety of other areas, such as, allergy, transplantation, and T-cell immunotherapies. 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. Human antibody fragments specific for human blood group antigens from a phage display library. The University of Chicago, Department of Biochemistry and Molecular Biology, 60637 Chicago, IL USA. SNAP-tagged targets: immobilization and specific proteolytic elution. Carmen S., Jermutus L. Concepts in antibody phage display. A. Rapid discovery and characterization of synthetic neutralizing antibodies against Anthrax Edema toxin. National Library of Medicine In addition, the GA1 fusions to the trans-membrane domains (TMDs) of cell receptors made in the endoplasmic reticulum, anchors the exchangeable Fab component to the eukaryotic cell surface, thus, enabling investigation of the cellcell signaling and interaction, and many other applications. Phage display as a promising approach for vaccine development Lfblom J. Bacterial display in combinatorial protein engineering. The novel approach developed in the KossLab yielded a rich pool of sAB, that could be used as crystallization chaperones, fiducial markers for SP cryo-EM or energy probes for different conformational states of a number of membrane proteins, laying foundation for elucidation of the high-resolution structures of the functionally relevant protein conformational states and understanding their dynamic interplay. 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. Multi-subunit proteins on the surface of filamentous phage: Methodologies for displaying antibody (Fab) heavy and light chains. Before Ahmadi M. K. B., Mohammadi S. A., Makvandi M., Mamouei M., Rahmati M., et al. PMC Nanodiscs, small (5-50 nm in diameter) discoidal particles consisting of lipids enclosed by the membrane scaffold proteins [133, 134] have been widely used in investigation of functional and structural properties of the membrane proteins as a sophisticated membrane mimetic system with precise control over their size and composition. Phage display offers a powerful approach to engineer protein affinity. Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display. The antibody-based fiducial markers have considerably aided the membrane-protein structural studies in the field of G-protein coupled receptor (GPCRs) [142-144]. Since these virions display the phagemid-encoded fusion protein, it provides physical link between the phage genotype and phenotype, realizing the key principle for phage-display as a molecular evolution-directed system [53]. Lokareddy R. K., Ko Y. H., Hong N., Doll S. G., Paduch M., et al. Bi-Fab is colored as following: GA1 is shown in red; fused to it, FabH (specific for HER2 receptor of cancer cell) in green, and FabLRT, specific to CD3 in blue. A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants. Compilation of the T-cell activation effects is shown. A novel -lactamase complementation-based assay. Diversity of the natural libraries is provided by the antibody repertoire acquired in the live immune system by gene rearrangement [12]. qpix, octet systems, pro, phage display, phage display library, antibody discovery, qpix Created Date: 9/30/2020 5:22:28 PM . Unauthorized use of these marks is strictly prohibited. Development of a Phage Display Panning Strategy Utilizing - Nature Bouvet J. P. Immunoglobulin Fab fragment-binding proteins. They have efficiently facilitated structure-determination of RNAs, RNAprotein complexes, protein complexes as well as individual proteins inherently recalcitrant to generate stable crystal lattices [83, 84, 130]. Real diversity of the nave human antibody repertoire is estimated to be at least 1012 unique combinations. To date, hundreds of different antibody-based formats have been engineered for therapeutic purposes, with many representing BiTEs [30], and new constructs are constantly emerging [31]. Protein and Antibody Engineering by Phage Display - PubMed Brogan A. P. S., Heldman N., Hallett J. P., Belcher A. M. Thermally robust solvent-free biofluids of M13 bacteriophage engineered for high compatibility with anhydrous ionic liquids. In 2017 the Nobel Prize in Chemistry was awarded jointly to Jacques Dubochet, Joachim Frank, and Richard Henderson for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution thus highlighting this technology as one of the greatest benefits to humankind [135]. coli filamentous bacteriophages (f1, fd, M13) are commonly used for phage display. If improvement of the sAB dissociation rate is at question, the possible trick would be prolongation of the wash-time. Comb Chem High Throughput Screen. Newton-Northup J. R., Dickerson M. T., Kumar S. R., Smith G. P., Quinn T. P., et al. This article does not contain studies with human participants or animals performed by the author. In addition, it is important to note, that the control over the linker length in the GA1 fusions could be an essential variable for applications in the systems with specific dimensional or steric demands. Additionally, fusion proteins could be rejected after the synthesis at different stages of phage maturation. The BiTE (bispecific T-cell engager) platform: Development and future potential of a targeted immuno-oncology therapy across tumor types. Although the biotinstreptavidin binding is not covalent, it serves all intents and purposes in biopanning, since the binding is almost irreversible (KD 1015 M). Almagro J. C., Pedraza-Escalona M., Arrieta H. I., Perez-Tapia S. M. Phage display libraries for antibody therapeutic discovery and development. 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. Shao Z., Arnold F. H. Engineering new functions and altering existing functions. To that end, an engineered variant of the apocytochrome b562, 12 kDa protein BRIL has been chosen as such a tag since it contains terminal helical extensions easily adjustable for the distortion-free seamless connection to -helices present either in the loops [145] or at the termini [146, 147] of the membrane proteins. It turned out that the Fab-binding affinity maturation has been beneficial for GA1 in other aspects as well: it significantly reduced Fc binding and practically abolished GA1 affinity to all natural Fabs tested, including human kappa (FabH) the parental to FabS [123].

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