Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity |
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Authors: | Hye Young Yang Kyung Jae Kang Julia Eunyoung Chung Hyunbo Shim |
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Institution: | (1) Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, 120-750, Korea;(2) Department of Life Science, Ewha Womans University, Seoul, 120-750, Korea |
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Abstract: | Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for
therapeutic and other applications. Antibody 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 β-lactamase selection strategy
was employed in which the assembled scFv gene repertoire was fused to the 5′-end of the β-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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Keywords: | Beta-lactamase selection functional diversity phage display scFv Synthetic antibody library |
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