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KMID : 0578320090270020225
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Molecules and Cells
2009 Volume.27 No. 2 p.225 ~ p.235
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Construction of a Large Synthetic Human scFv Library with Six Diversified CDRs and High Functional Diversity
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Yang Hye-Young
Kang Kyung-Jae
Chung Eunyoung Julia
Shim Hyun-Bo
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Abstract
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Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Anti-body clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a 棺-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5??end of the 棺-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 ??109, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.
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KEYWORD
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Beta-lactamase selection, functional diversity, phage display, scFv, Synthetic antibody library
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