SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping

Nucleic Acids Res. 2019 Apr 8;47(6):e34. doi: 10.1093/nar/gkz050.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Peptide Library
  • Protein Engineering / methods*
  • Single-Chain Antibodies / genetics
  • Solid-Phase Synthesis Techniques / methods*
  • Staphylococcus / genetics
  • Synthetic Biology / methods
  • Uracil / chemistry
  • Uracil / metabolism*

Substances

  • Mutant Proteins
  • Peptide Library
  • Single-Chain Antibodies
  • Uracil
  • DNA Restriction Enzymes