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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
The phage display libraries were able to present peptides not only in linear but also in circular form, when the epitope sequence was flanked by two cysteine residues. By building a disulfide bond, a constrained cycle was formed and presented on the phage surface (Luzzago et al. 1993). Nevertheless, the length of the inserted peptides did not exceed 6–38 aa residues, as reviewed by Hill and Stockley (1996). To overcome the capacity problem, the phagemid systems were developed, where the phagemid carried only the phage gene g3 or g8 containing the foreign sequence, and a phage with all the necessary genes for phage production, also including a copy of the wild-type coat gene, was used as a helper. Thus, both chimeric and wild-type proteins were produced and incorporated into the mosaic virions, when one of five copies of p3 and 1% to 30% of the 2700 copies of p8 were chimeric, as reviewed by Davies et al. (2000).
Molecular Farming Antibodies in Plants: From Antibody Engineering to Antibody Production
Published in Maurizio Zanetti, J. Donald Capra, The Antibodies, 2002
Rainer Fischer, Ricarda Finnern, Olga Artsaenko, Stefan Schillberg
Both phage [44] and phagemid [18-21] vectors have been used for the display of antibody fragments but, generally, phage vectors have been superseded by phagemids. Phagemid vectors are small plasmids that replicate in bacteria but are incapable of forming phage particles without the addition of helper phage. Phagemids have high bacterial transformation efficiencies, are ideally suited to cloning of large repertoires, and can be formatted to direct the secretion of soluble antibody fragments without subcloning [18]. The phagemid system is based on a vector containing only the genes encoding the gene III or gene VIII proteins and bacterial and viral origins of replication. To produce phage particles, bacterial cells containing the phagemid are rescued with helper phage strains that provide all the remaining proteins needed for the generation of phage [45]. Replication of the helper phage DNA is less efficient than that of the phagemid and therefore only phagemid DNA is packed into the phage particle. However, since helper phage encodes wild-type gene III and its product (g3p), over 90% of the rescued phagemid particles display no antibody. This is a consideration when selecting large antibody libraries if all encoded antibodies in the library are to be represented. Equally important, most phagemid particles displaying antibody
Improved therapeutic index of an acidic pH-selective antibody
Published in mAbs, 2022
Peter S. Lee, Katherine G. MacDonald, Evan Massi, Pamela V. Chew, Christine Bee, Padma Perkins, Bryant Chau, Kent Thudium, Jack Lohre, Pradyot Nandi, Ekaterina G. Deyanova, Ishita Barman, Olafur Gudmundsson, Gavin Dollinger, Tim Sproul, John J. Engelhardt, Pavel Strop, Arvind Rajpal
The ipilimumab-CTLA-4 epitope and paratope were identified from the available complex crystal structure (PDB ID 5TRU) using CCP4 programs42 Contact and AreaiMol and were then manually inspected. An initial mini library was built by splicing by overlap extension polymerase-chain reaction (SOE-PCR). One histidine substitution introduced into each heavy chain CDR was enforced at positions 28, 30, 31, 32, 33, 52, 52a, 53, 54, 55, 56, 95, 97, and 98 (Kabat numbering) for a total of three histidine mutations per variant, resulting in a theoretical diversity of 90 members. The single-chain variable fragment (scFv) library was inserted by Gibson Assembly (NEB) into the phagemid vector, which contains a phoA promoter, pelB leader sequence, and a C-terminal FLAG tag followed by a fragment of the phage coat protein pIII. The phagemid DNA was electroporated into SS320 cells (Lucigen) and the phage library was propagated and purified.
SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology
Published in mAbs, 2023
Sarah-Jane Kellmann, Christian Hentrich, Mateusz Putyrski, Hanh Hanuschka, Manuel Cavada, Achim Knappik, Francisco Ylera
The in vitro selection of peptides and proteins with desired properties from large gene libraries is a powerful approach that has been used extensively for the discovery of binding molecules, including antibodies.1,2 All in vitro selection technologies require the physical linkage of genotype and phenotype. Phage display is the oldest and most widely used method due to its robustness and favorable properties, such as speed, simplicity, and accommodation of large libraries. To couple genotype to phenotype, the proteins to be selected are displayed on the surface of engineered filamentous M13 phages, which contain the genetic information of the presented proteins. In conventional phage display, the displayed protein is genetically fused to a coat protein of the phage, in most cases to the minor coat protein pIII. Instead of a full phage genome, smaller plasmid derivatives called phagemids are commonly used in phage display, as they simplify cloning and allow the creation of larger libraries.3,4 Such phagemids contain the genes for the displayed proteins fused to the gene encoding the phage coat protein used for display, antibiotic resistance genes, and genetic elements required for plasmid-like replication as well as for replication of ssDNA and its packaging in phage capsid. Phage display with phagemids necessitates the use of helper phage, which provides the remaining structural and regulatory phage proteins that are not present in the phagemid and thus allows phage assembly after superinfection of phagemid-containing bacteria. After several rounds of phage display, the selected genes are typically subcloned into a suitable expression plasmid for screening and further analysis.5
Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application
Published in Drug Delivery, 2021
Nattihda Parakasikron, Chatchai Chaotham, Pithi Chanvorachote, Chanida Vinayanuwattikun, Visarut Buranasudja, Pornchanok Taweecheep, Kannika Khantasup
The phagemid from each positive clone was extracted to determine the DNA sequence of the VH in the recombinant phagemid. The VH sequencing was performed using the pR2-vector specific primers LMB3: 5′-CAGGAAACAGCTATGAC-3′. The DNA sequences and the deduced amino acid sequences were compared with the DNA sequences in the GenBank sequence database to determine the complementarity-determining regions (CDR) and framework.