Cassette Hybridization for Vector Assembly Application in Antibody Chain Shuffling

Lai, Jing Yi; Loh, Qiuting; Choong, Yee Siew; Lim, Theam Soon

doi:10.2144/btn-2018-0031

Cited by 3 publications

(2 citation statements)

References 25 publications

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“…In all cases, a protein linker (typically 15–20 amino acids long) is used to connect the two immunoglobulin domains. Strategies for generating site-directed scFv libraries usually rely on primer extension, gene assembly, recombination or single-stranded templates to generate the necessary genetic diversity ( 14 , 22 , 24 , 25 ).…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template

et al. 2021

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Directed DNA libraries are useful because they focus genetic diversity in the most important regions within a sequence. Ideally, all sequences in such libraries should appear with the same frequency and there should be no significant background from the starting sequence. These properties maximize the number of different sequences that can be screened. Described herein is a method termed SLUPT (Synthesis of Libraries via a dU-containing PCR Template) for generating highly targeted DNA libraries and/or multi-site mutations wherein the altered bases may be widely distributed within a target sequence. This method is highly efficient and modular. Moreover, multiple distinct sites, each with one or more base changes, can be altered in a single reaction. There is very low background from the starting sequence, and SLUPT libraries have similar representation of each base at the positions selected for variation. The SLUPT method utilizes a single stranded dU-containing DNA template that is made by PCR. Synthesis of the template in this way is significantly easier than has been described earlier. A series of oligonucleotide primers that are homologous to the template and encode the desired genetic diversity are extended and ligated in a single reaction to form the mutated product sequence or library. After selective inactivation of the template, only the product library is amplified. There are no restrictions on the spacing of the mutagenic primers except that they cannot overlap.

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Section: Resultsmentioning

confidence: 99%

Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template

et al. 2021

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“…The gene cassettes were initially prepared by PCR amplification, followed by vector assembly with the desired gene combinations through hybridization using solely DNA polymerase at optimized conditions. The chain-shuffling method yielded a comparable outcome with respect to the conventional restriction digestion and ligation method [190].…”

Section: In Vitro Affinity Maturation Strategiesmentioning

confidence: 99%

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Lim

Choong

Lim

2019

IJMS

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Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.

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Antibody display technologies: selecting the cream of the crop

Valldorf

Hinz

Russo

et al. 2021

Biological Chemistry

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Antibody display technologies enable the successful isolation of antigen-specific antibodies with therapeutic potential. The key feature that facilitates the selection of an antibody with prescribed properties is the coupling of the protein variant to its genetic information and is referred to as genotype phenotype coupling. There are several different platform technologies based on prokaryotic organisms as well as strategies employing higher eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic antibodies approved for therapy that have been discovered or engineered using this approach. In recent years several other technologies gained a certain level of maturity, most strikingly mammalian display. In this review, we delineate the most important selection systems with respect to antibody generation with an emphasis on recent developments.

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Cassette Hybridization for Vector Assembly Application in Antibody Chain Shuffling

Cited by 3 publications

References 25 publications

Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template

Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Antibody display technologies: selecting the cream of the crop

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