SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping.
Antibody Affinity
/ genetics
Binding Sites, Antibody
/ genetics
Cloning, Molecular
/ methods
DNA Restriction Enzymes
/ metabolism
Epitope Mapping
High-Throughput Nucleotide Sequencing
Mutagenesis, Site-Directed
/ methods
Mutant Proteins
/ chemistry
Peptide Library
Protein Engineering
/ methods
Single-Chain Antibodies
/ genetics
Solid-Phase Synthesis Techniques
/ methods
Staphylococcus
/ genetics
Synthetic Biology
/ methods
Uracil
/ chemistry
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
08 04 2019
08 04 2019
Historique:
accepted:
24
01
2019
revised:
08
01
2019
received:
28
09
2018
pubmed:
5
2
2019
medline:
13
11
2019
entrez:
5
2
2019
Statut:
ppublish
Résumé
Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.
Identifiants
pubmed: 30715449
pii: 5304719
doi: 10.1093/nar/gkz050
pmc: PMC6451119
doi:
Substances chimiques
Mutant Proteins
0
Peptide Library
0
Single-Chain Antibodies
0
Uracil
56HH86ZVCT
DNA Restriction Enzymes
EC 3.1.21.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e34Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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