phage display system for protein engineering

Recent advances in the scaffold engineering of protein binders. Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. This vector, called a phagemid, can be transformed and amplified in E. coli as a regular plasmid. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the . Samish I. Moore J. C., Arnold F. H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. This mostly releases the target-bound phage limiting selection of the non-specific clones [112, 113], but requires additional amounts of the purified antigen. Silacci M., Brack S., Schirru G., Mrlind J., Ettorre A., 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]. However, the M13 phage has its own pluses, making it the most developed and popular antibody-display platform to date. Miller E. A., Sung K. J., Kongsuphol P., Baniya S., Aw-Yong H. Q., et al. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. 2016;22(43):6538-6559. doi: 10.2174/1381612822666160923113714. Each of these proteins has a different antibody binding profile in terms of the portion of the antibody that is recognized and the species and type of antibodies it binds. 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). A novel -lactamase complementation-based assay. The principle underlying all phage display systems is the physical linkage of a polypeptide's phenotype to its corresponding genotype. A. 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. The KossLab pipeline production also have confirmed an undeniable potential of sABs targeting different antigen determinants in translational medicine. In this system, it is necessary that the protein is displayed on the . Several selected variants demonstrated significantly increased binding affinity and reduced dissociation rate. qpix, octet systems, pro, phage display, phage display library, antibody discovery, qpix Created Date: 9/30/2020 5:22:28 PM . The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Innovations 6 , 1-6 (1996). Bookshelf Farcasanu M., Wang A. G., Uchaski T., Bailey L. J., Yue J., et al. Rizk S. S., Mukherjee S., Koide A., Koide S., Kossiakoff A. Adv Sci (Weinh). Protein posttranslational modifications: the chemistry of proteome diversifications. 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. The KossLab pipeline have been constructed based on the scaffold from a humanized Fab 4D5 Herceptin framework (here, FabS) engineered for high stability and efficient phage display. Synthetic antibodies for specific recognition and crystallization of structured RNA. As mentioned above, we have developed a wash-free antigen-detection system based on the GA1 module fusions in a protein-fragment complementation format, widely used for identification and studying of proteinprotein interactions. As discussed above, the pre-existing variety of CDRs incorporated into the natural antibody libraries originates from the operational immune system in vivo [12], therefore, quality of the natural library depends, mostly, on the efficiency of the variety incorporation into the library, i.e., on the library size. Since then, phage display has become a molecular powerful tool for selection of peptide/antibody fragments with specific binding properties from a huge number of variants (libraries) by presenting such fragments on the phage surface, and therefore generating molecular probes against specific targets (Willats 2002 ). Proteins A, G, and L have a scope of biochemical, biotechnological, and medical applications and are widely used in antibody purification and test methods like immunoprecipitation, ELISA, and Western blotting [153]. Both scFvs and Fabs have been displayed on the bacteriophage surface in the Winters pioneering works [24, 36] and are still the most popular for construction of sAB phage display libraries. Dean A. Q., Luo S., Twomey J. D., Zhang B. 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]. Kang Y., Kuybeda O., de Waal P. W., Mukherjee S., Van Eps N., et al. 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]. Human antibody fragments specific for human blood group antigens from a phage display library. Li T, Cai H, Zhao Y, Li Y, Lai Y, Yao H, Liu LD, Sun Z, van Vlissingen MF, Kuiken T, GeurtsvanKessel CH, Zhang N, Zhou B, Lu L, Gong Y, Qin W, Mondal M, Duan B, Xu S, Richard AS, Raoul H, Chen J, Xu C, Wu L, Zhou H, Huang Z, Zhang X, Li J, Wang Y, Bi Y, Rockx B, Chen J, Meng FL, Lavillette D, Li D. EMBO Mol Med. Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display. Phage display has become established as a powerful protein engineering method for identifying polypeptides with novel properties, and altering the properties of existing ones. However, binding is still characterized by fast dissociation kinetics that is not optimal for the desired non-equilibrium applications. An engineered FABLRT As mentioned above, principal distinction between the phage display and directed evolution is that DNA does not undergo further diversification between the rounds of phage display panning. Screening of novel peptides that specifically interact with vitamin D bound biocomplex proteins. An engineered protein tag for multiprotein labeling in living cells. Therefore, smart diversification strategy considering each CDR length variability and randomization method as well as their magnitude became the key issue in the design of a quality sAB library [69]. 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. Denisov I. G., Sligar S. G. Nanodiscs for structural and functional studies of membrane proteins. Although the biotinstreptavidin binding is not covalent, it serves all intents and purposes in biopanning, since the binding is almost irreversible (KD 1015 M). As expected, functionality of the bi-Fabs was absolutely dependent upon the genetic fusion of GA1 to FabH, as no detectable activity was observed when all the individual components (FabH, FabLRT, GA1) were added as three separate unlinked entities [123]. Costa S. J., Coelho E., Franco L., Almeida A., Castro A., et al. Respective modulations of the MBP ligand-binding affinity in competitive, allosteric, or peristeric manners, were used as probes to quantify energy contributions of the ligand binding to the conformational changes in proteins. Depending on the antigen immobilization technique, elution methods also vary widely. sABs as tools for protein structure determination. Baeuerle P. A., Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. This modification level is sufficient for efficient capture of the antigen on the streptavidin-coated microplates or streptavidin-coupled magnetic beads followed by the library panning. The https:// ensures that you are connecting to the Caberoy N. B., Zhou Y., Jiang X., Alvarado G., Li W. Efficient identification of tubby-binding proteins by an improved system of T7 phage display. 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. Architecture of the nuclear pore complex coat. Federal government websites often end in .gov or .mil. Synthetic antibodies, tailored to a specific antigen or antigen epitope hugely helped to overcome protein structure determination challenges as well as powered up many medical endeavors. 4e) with the interchangeable FabLRT specificity that could be utilized in place of multiple IgGs in the Fc-mediated processes. 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. In addition to phage display, a great variety of display platforms have been developed to better suit rationale and particularity of the selections. Success of complementation and enzyme reactivation depend to a great extent on the appropriateness between the length of fusion linkers and the distance between the antigen epitopes targeted by Fabs. Van den Brulle J., Fischer M., Langmann T., Horn G., Waldmann T., et al. 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. You L., Arnold F. H. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Some nave antibody libraries represent 1011 individual clones or more [18, 21, 22], reaching practical limit of the phage antibody library size preset around 1012 value, due to the technical limitation of bacterial transformation, culturing, and storage [62]. Targeting cancer with antibody-drug conjugates: Promises and challenges. sAB customization strategies. Immobilization of the SNAP-tagged target may be achieved by means of either direct covalent attachment to SNAP-capture beads or by biotinylation of SNAP-tagged target by SNAP-biotin prior to the Streptavidin-coupled bead binding. Protein antigen could be chemically conjugated with biotin [103] using commercially available biotinylation reagents targeting variety of specific functional groups or residues, including primary amines, sulfhydryls, carboxyls, and carbohydrates [104]. The modified library-panning protocol developed in the KossLab for the SNAP-tagged targets (Fig. doi: 10.1016/j.chembiol.2022.02.004. Recently, we developed a novel phage display system using the coliphage Q as a nano-biotechnology platform. Inclusion in an NLM database does not imply endorsement of, or agreement with, Single-stranded DNA with, Kunkel mutagenesis. Weinstein JB, Bates TA, Leier HC, McBride SK, Barklis E, Tafesse FG. Yang H. Y., Kang K. J., Chung J. E., Shim H. Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. These chains, of approximately equal masses, comprise ~50 kDa Fab. A novel plug-and-play BI-Fab format. Phage display was furthermore applied for improving the catalytic activity of enzymes by in vitro evolution (Soumillion et al., 1994; Pedersen et al., 1998). Alternatively, a specific conformational state of an antigen could be targeted already at the design conception. A novel wash-free -lactamase complementation-based detection assay (Fig. Also, sAB solubility, was addressed in the design of shotgun scanning libraries introducing aspartate as a negative design element at the antigen-binding site. 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. and transmitted securely. This gp5 shell packs genomic DNA into a flexible rod protected from nucleases, thus forming the intracellular precursor of the extracellular virion. K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle. 8600 Rockville Pike A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants. 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. 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. A. Epitope binning revealed that multiple distinct epitopes were targeted for each of trimeric G-protein, while some of the sABs cross-react between the trimeric Gi and Gs, suggesting their universal application across the subclasses. Compared to the wild-type protein G, GA1 demonstrates a significant affinity boost in binding FabS (KD ~ 50 nM) adequate for applications that involve genetically linked GA1 strings to make multi-valent Fab assemblages [157]. The scFv molecule is half a size (~25 kDa) and consists of two variable domains (VH and VL) fused together by a small flexible peptide linker of ~15 aa (Fig. Introduction and potential of therapeutic applications. This article does not contain studies with human participants or animals performed by the author. A. Phage display technology: clinical applications and recent innovations. The phage display for protein engineering, and other applications such as peptide or whole-phage engineering are described. To construct bi-Fabs in either polarities, each antibody was reformatted in both FabH and FabLRT scaffolds and all three resultant FabH variants were fused to GA1 at their Hc C-termini via a 13 aa-long GS linker. The key structural basis underlying this ultra-high affinity of the FabLRT was revealed in a crystal structure solved for the GA1FabLRT complex: a significant deletion-induced rearrangement and extension of the interface has packed the guanidinium group of Arg124 (replacing Lys126 in FabS) against the aromatic ring of Tyr40 of GA1 in a cation interaction, while providing H-bonding with the Tyr40 carbonyl via the secondary amine in position of Arg124 [123]. Due to the simpler design, these libraries are relatively easy to construct, and, due to the smaller theoretical size, they possess higher sampling power. Shao Y., Huang H., Qin D., Li N. S., Koide A., et al. The Fh8 tag: a fusion partner for simple and cost-effective protein purification in. Walsh C. T., Garneau-Tsodikova S., Gatto G. J., Jr. There are hundreds of different GPCRs belonging to 3 major classes encoded in human genome. Almagro J. C., Pedraza-Escalona M., Arrieta H. I., Perez-Tapia S. M. Phage display libraries for antibody therapeutic discovery and development. 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]. The LVPRGS thrombin-cleavage site in the linker between the target and SNAP-tag is shown. The most common affinity-independent elution method entails insertion of a unique proteolytic site next to the affinity tag [114]. Another important aspect defining functionality of the sAB phage display is a balance between the theoretical number of possible variants set by the design, and the number of unique phage clones achieved at a construction step, i.e., the library sampling power. They play a central physiological role in the regulation of cellular responses to a wide variety of stimuli in both health and disease and thus represent one of the largest types of surface receptors targeted by drugs. Tiller T., Schuster I., Deppe D., Siegers K., Strohner R., et al. The .gov means its official. Under such conditions, generation of a conformationally-selective antibody recognizing just one of the antigen states is accidental. The selected high-affinity variants featured a polar ring surrounding the paratope. 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). M13 phage has high capacity for replication and can accommodate large foreign DNA as a highly transformable M13-based dsDNA phagemid vector, mimicking phage replicative form (RF) and producing circular ssDNA phagemid molecules that can be packaged into infectious phage particles. Schaefer Z. P., Bailey L. J., Kossiakoff A. Structure of the micro-opioid receptorGi protein complex. 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]. The detection limit of the systems (~10 nM) falls within the standard range for a sandwich antigen-detection immunoassay, like ELISA. Three of them: L1, L2, and L3 are located in VL, and three: H1, H2, H3 in VH. Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling. High specificity and orthogonal nature of the GA1FabLRT binding also allowed us to genetically connect a second specificity to the GA1 module in a form of FabH, or scFv, without being concerned of their possible interaction and interference with the FabLRT binding. In addition, phage elution of any kind can be skipped, if no phage tittering is needed, instead, E. coli cells could be directly infected with the antigenbound phage in selection wells or by addition of the phage pulled out on magnetic beads. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. 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. The CDR loops of a sAB library need to be designed so that the resulting library is enriched with diverse, yet nature-like sequences, different in length and composition [70]. Therefore, smaller, mono-valent antigen-binding fragments such as scFv (single chain fragment variable) or Fab (fragment antigen-binding) are the most often used formats for phage display [32] (Fig. Passariello M., Gentile C., Ferrucci V., Sasso E., Vetrei C., et al. Activation of the CD8 cytotoxic T-cell involves cytolytic granule fusion and release directed toward the cancer cell, while the activated CD4 helper T-cells increase production and secretion of cytokines interferon and IL2, activating cytokine production, cytolytic effect, and proliferation of peripheral blood immune cells. A new fusion protein platform for quantitatively measuring activity of multiple proteases. England C. G., Luo H., Cai W. HaloTag technology: a versatile platform for biomedical applications. On the contrary, theoretical number of the possible combinations in the case of synthetic CDR variants has almost no upper limit: e.g., complete randomization of just a single piece of 10 codons produces more than 1013 (2010 including stop codons) of clone variations amount, practically unattainable in a phage library. The resulting bi-Fabs, functionally mimicking the bi-specific T-cell engagers (BiTEs), very efficient immunotherapeutics, have the advantage of one easily interchangeable specificity and are discussed in more details below. 4a). Engineered proteins as specific binding reagents. Rizk S. S., Paduch M., Heithaus J. H., Duguid E. M., Sandstrom A., et al. 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]. Eliasson M., Olsson A., Palmcrantz E., Wiberg K., Ingans M., et al. Hinner M. J., Johnsson K. How to obtain labeled proteins and what to do with them. These sABs stabilize MBP in different conformational states as has been revealed in the respective crystal structures. A. Dominik P. K., Borowska M. T., Dalmas O., Kim S. S., Perozo E., et al. Azzazy H. M., Highsmith W. E., Jr. Phage display technology: clinical applications and recent innovations. Zheng S., Sham L. T., Rubino F. A., Brock K. P., Robins W. P., et al. In fact, an unfavourable selection pressure during . SNAP-tag has been engineered to reacts with O(6)-benzylguanine, while CLIP-tag with O(2)-benzylcytosine derivatives. FOIA Specific recognition of a single-stranded RNA sequence by a synthetic antibody fragment. Protein A has greater affinity for the rabbit, pig, dog, and cat IgG whereas protein G has greater affinity for the mouse and human IgG. These would eliminate the need to incorporate the challenge of an antibody-engineering into the workflow of protein-structure determination projects. See this image and copyright information in PMC. Hussack G., Baral T. N., Baardsnes J., van Faassen H., Raphael S., et al. Protein engineering is now a mature field of protein science. By driving a protein or its flexible parts to adopt and halt a uniform conformation, the conformation-specific sABs can enable crystallization of unstable, multi-conformational, multi-component or multi-subunit structures, membrane proteins and their complexes [81, 82, 98, 131]. The recent world threats caused by severe viral infections motivated us to choose Ebola and Zika virus proteins (EBOV NTCT and MT ZIKV, respectively) as antigens to be detected in the system and used initially for generation of the high-affinity sABs by phage-display selections reformatted next into the FabLRT scaffold. GA1-based plug-and-play platforms. Next, the membrane-associated gp8 oligomers are built into a spiraling array around phage DNA, replacing gp5. 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. 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. Two different CD3-specific FabLRT, derivatives of OKT3 and UCHT1 antibodies, were interchangeably used with trastuzumab-derived FabH fused to GA1. 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. Winter G., Griffiths A. D., Hawkins R. E., Hoogenboom H. R. Making antibodies by phage display technology. 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. 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. Recognition of an alpha-helical hairpin in P22 large terminase by a synthetic antibody fragment. 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]. Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. The BiTE (bispecific T-cell engager) platform: Development and future potential of a targeted immuno-oncology therapy across tumor types. Bacteriophages M13, fd, and f1 belonging to Ff (F-pilus specific filamentous) phages and almost identical in every aspect (98% identity at the DNA level) were the first used in phage display [48]. Before Frank J. Cryo-electron microscopy as an investigative tool: The ribosome as an example. 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]. Antibody engineering; Combinatorial biochemistry; Combinatorial scanning mutagenesis; Phage display; Protein engineering. While theoretical diversity of the nave combinatorial libraries is much higher than their achievable size, there is significantly less variety of the naturally paired VH-VLs in the mammalian immune system. Comb Chem High Throughput Screen. 1) Target immobilization on magnetic beads. Nordenfelt P., Bjrck L. IgG-binding bacterial proteins and pathogenesis. Natural antibody libraries have been proven to be excellent suppliers of high-quality antibodies suitable for therapeutic applications in medicine [65] and veterinary [66], and many such antibodies are in clinical use or at various stages of therapy development [40]. 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. Unable to load your collection due to an error, Unable to load your delegates due to an error, Phage display selection. Housden N. G., Harrison S., Roberts S. E., Beckingham J. Einsele H., Borghaei H., Orlowski R. Z., Subklewe M., Roboz G. J., et al. Bass S., Greene R., Wells J. A., Rock R. S. Characterization of engineered actin binding proteins that control filament assembly and structure. doi: 10.1002/advs.202103645. Adler A. S., Mizrahi R. A., Spindler M. J., Adams M. S., Asensio M. A., et al. The site is secure. The antibody assortment can be enriched for the desired-target binders by prior immunization. 2002 Sep;35(6):425-45. doi: 10.1016/s0009-9120(02)00343-0. Nature of the coat protein to be used as the fusion partner as well as size and structural characteristics of the proteins chosen to be displayed should be considered for the display success. Membrane proteins are difficult to purify in a native form, and conditions matching their natural surroundings are hard to reproduce for crystallization purposes. The nanodisc-based native-like lipid environment and the overall configuration of the system allow the partially imbedded target protein to adopt characteristic structural transitions, and the sABs to bind to these transient conformations. A novel solid phase technology for high-throughput gene synthesis. (A) Serial dilutions of DENV-2 DIII-expressing phage were incubated with DIII-binding antibody 4E11 (. Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.

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