Integrated Mimicry of B Cell Antibody Mutagenesis Using Yeast Homologous Recombination
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nihms-565552.pdf
Description
Accepted version
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1.34 MB
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Author(s)
Swers, Jeffrey Seth
Yeung, Yik Andy
Wittrup, Karl Dane
Date Issued
January 2011
Journal
Molecular Biotechnology
Publisher
Springer Nature America, Inc
Citation
Swers, Jeffrey S., Yik A. Yeung, and K. Dane Wittrup. "Integrated Mimicry of B Cell Antibody Mutagenesis Using Yeast Homologous Recombination." Molecular Biotechnology 47,1 (January 2011):p.57-69. © Springer Science+Business Media, LLC 2010.
Version
Author's final manuscript
Abstract
Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mutagenesis. This method was used to effect a 10,000-fold affinity improvement in an anti-peptide single-chain antibody in three rounds of mutagenesis and screening, and a 1,000-fold affinity improvement in an anti-protein single-chain antibody in a single round. When recombinational mutagenesis (CDR or chain shuffling) was directly compared to error-prone PCR, the recombinational approach yielded greater affinity improvement with substantially reduced divergence from germline sequences, demonstrating an advantage of simultaneously testing a broad range of mutational strategies. Keywords: east display; Affinity maturation; Homologous recombination; Receptor editing; Receptor revision; Chain shuffling; Antibody engineering; Library design
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1007/s12033-010-9312-3
