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Order Tubulavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Juds et al. (2020) demonstrated how to combine the phage display with next-generation sequencing (NGS) for the materials sciences by a study on probing polypropylene. Thus, the phage display biopanning with Illumina NGS was applied to reveal insights into the peptide-based adhesion domains for polypropylene. Remarkably, the single biopanning round followed by NGS selected robust polypropylene-binding peptides that were not evident through Sanger sequencing. The NGS provided a significant statistical base that enabled motif analysis, statistics on positional residue depletion/enrichment, and data analysis to suppress false-positive sequences from amplification bias (Juds et al. 2020).
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Affibodies are the small protein fragments obtained from immunoglobulin binding region B-domain (~58 amino acids) of staphylococcal protein A. Structurally, affibodies are folded in a three-helix bundle and it has been reported that they follow the fastest folding kinetics. B-domain is thermally very stable. To increase its chemical stability, some of the residues were mutated, which resulted in the formation of “Z-domain.” The resulting Z-domain maintained its affinity for Fc part of antibody but lost its affinity for Fab part of the antibody (Nilsson et al., 1987). Affibody molecules with different specificities were designed using the randomization of 13 amino acids in the first and second helices that constitute the Fc binding surface of Z-domain. Affibody molecules for specific protein targets were then screened using biopanning technique (Nord et al., 1997). Affibody molecules specific to different proteins have been engineered that include IL-8, CD28, transferrin, tumor necrosis factor-a, fibrinogen insulin, gp-120, EGFR, HER2, IgA, IgM, IgE, and human serum albumin. Affibody molecules engineered for all these proteins showed binding affinity ranging from μM to pM range. Affibody molecules due to their small size can also be fused to other molecules and can still maintain their size smaller than the conventional antibody (Nygren, 2008; Lofblom et al., 2010).
Recombinant Antibodies
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Melvyn Little, Sergey M. Kipriyanov
In view of the paracrystalline array of the major coat protein pVIII and the lack of pVIII mutants (Kuhn and Wickner, 1985), it is surprising that several hundred peptides fused to the N-terminus of pVIII can be incorporated into the phage coat without preventing phage assembly (Felici et al., 1991; Ilyichev et al., 1992). Larger polypeptides, however, do not appear to be readily incorporated. Kanget al. (1991a) reported that from one to twenty Fd fragments (heavy chain variable domain plus the first constant domain) fused to pVIII were incorporated along the length of the phagemid particle. Free Fab fragments can be generated, as described above for pIII fusions, by incorporating an amber mutation between the antibody and p VIII domains (Huse et al., 1992). Although the pVIII systems proved to be useful for isolating antibodies with low affinities, they are less efficient than the pIII system for screening antibody libraries (Gram et al., 1992). Direct comparison of pIII and pVIII systems demonstrated a much higher display efficiency of scFv fused to pIII on the phage surface as judged by ELISA. Furthermore, more scFv::pIII than scFv::pVIII phage could be recovered in biopanning experiments (Kretzschmar and Geiser, 1995).
Phage in cancer treatment – Biology of therapeutic phage and screening of tumor targeting peptide
Published in Expert Opinion on Drug Delivery, 2022
Arun Chandra Manivannan, Ranjithkumar Dhandapani, Palanivel Velmurugan, Sathiamoorthi Thangavelu, Ragul Paramasivam, Latha Ragunathan, Muthupandian Saravanan
Despite the pioneering works of George Smith and the patent of George Pieczenick (US patent, 5866363), 2018 chemistry Noble Prize winners Smith and Winter contributed significantly to their decade-long research in experimenting with various display techniques. Biopanning is a widely used five-step selection technique. The stages are as follows: a) Library construction and amplification; b) Target capturing step; c) Removal of unbound and nonspecific phages; d) Elution, and finally; e) Infection stage. This cycle is repeated around three to five rounds to isolate high-affinity peptide binders. ELISA and DNA sequencing are terminal steps to identify specific high-affinity pages [58]. In vivo biopanning involves the injection of phage to live host animals like lab rats, while in vitro involves infecting the cell line grown in labs. Ex vivo involves fewer studies but is still a sound method of biopanning [59].
Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy
Published in Expert Opinion on Biological Therapy, 2021
Anne-Fleur E. Schneider, Annemieke Aartsma-Rus
While CPPs improve delivery in general, they do not prevent that most AONs are taken up by liver and kidney. Conjugating a ligand to the AONs that directs them to skeletal muscle and heart is an obvious way to increase delivery. However, the challenge lies in finding these peptides. One way to achieve this is using phage display libraries and biopanning experiments. Here a library of phages, each displaying a unique peptide on their surface is incubated with a target molecule, cell cultures, or injected into an animal model. Generally, libraries are enriched with multiple biopanning rounds, after which the library is sequenced and overrepresented peptides are selected as molecule or tissue-specific peptides. Using this approach, a number of muscle and heart homing peptides have been identified (Table 3).
Selection of target-binding proteins from the information of weakly enriched phage display libraries by deep sequencing and machine learning
Published in mAbs, 2023
Tomoyuki Ito, Thuy Duong Nguyen, Yutaka Saito, Yoichi Kurumida, Hikaru Nakazawa, Sakiya Kawada, Hafumi Nishi, Koji Tsuda, Tomoshi Kameda, Mitsuo Umetsu
The biopanning procedure was described previously.34 Briefly, N11–N14 and M66–K72 in 2u2f were randomized using degenerate codons reflecting an amino acid frequency of antibody CDRs37 for training data. M13 phage libraries displaying 2u2f variants with a size of ~109 were prepared. Colony-forming units (5.0 × 1011) from an M13 phage library displaying 2u2f variants were exposed to magnetic beads (Dynabeads MyOne Streptavidin T1 or C1; Thermo Fisher Scientific, MA, USA) for 60 min at room temperature (Negative selection in Figure 2). For target preparation, 2 µM galectin-3 in PBS was incubated with magnetic beads for 60 min at room temperature such that the amount of targets on beads was 9 µg, which was calculated from the amount of supernatants measured by means of BCA assay using bovine serum albumin as a standard (Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific, MA, USA). The supernatant containing unbound phages was collected and incubated with galectin-3–immobilized magnetic beads for 60 min at room temperature. The beads were washed 10 times with PBS with 0.05% Tween-20 for 5 min each wash. Bound phages were eluted with 100 µL of triethylamine and neutralized with 300 µL of 1 M Tris–HCl (pH 6.8). Log-phase E. coli TG-1 cells were incubated overnight at 37°C with 200 µL of the eluted phages in 2× YT agar medium containing 100 µg/mL ampicillin and 1% (w/v) glucose. Cells grown on the plates were used to prepare phage particles for the next round. For the training data, phage pools were collected at each step (eluted phages, infected E. coli, and amplified phages) for deep sequencing analysis.