In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region
Autor: | Daniel Cannon, Bojana Popovic, D. Gareth Rees, Ralph Minter, Andrew Buchanan, Kalliopi Skamaki, Stéphane Emond, John Andrews, Florian Hollfelder, Matthieu Chodorge |
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Rok vydání: | 2020 |
Předmět: |
Transposable element
Antibody Affinity Immunoglobulin Variable Region Mutagenesis (molecular biology technique) Computational biology Biology 010402 general chemistry 01 natural sciences Antibodies Evolution Molecular 03 medical and health sciences INDEL Mutation Humans Indel Sequence Deletion 030304 developmental biology 0303 health sciences Multidisciplinary Point mutation food and beverages Protein engineering Biological Sciences Directed evolution 0104 chemical sciences Deletion Mutagenesis Mutagenesis Insertional Mutagenesis Ribosome display Genetic Engineering |
Zdroj: | Proc Natl Acad Sci U S A Proceedings of the National Academy of Sciences |
ISSN: | 1091-6490 0027-8424 |
Popis: | We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity. |
Databáze: | OpenAIRE |
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