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SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping.
Hu, Francis Jingxin; Lundqvist, Magnus; Uhlén, Mathias; Rockberg, Johan.
Afiliação
  • Hu FJ; KTH - Royal Institute of Technology, Department of Protein Science, 106 91 Stockholm, Sweden.
  • Lundqvist M; KTH - Royal Institute of Technology, Department of Protein Science, 106 91 Stockholm, Sweden.
  • Uhlén M; KTH - Royal Institute of Technology, Department of Protein Science, 106 91 Stockholm, Sweden.
  • Rockberg J; KTH - Royal Institute of Technology, Science for Life Laboratory, Solna 171 65, Sweden.
Nucleic Acids Res ; 47(6): e34, 2019 04 08.
Article em En | MEDLINE | ID: mdl-30715449
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Uracila / Sítios de Ligação de Anticorpos / Engenharia de Proteínas / Mutagênese Sítio-Dirigida / Técnicas de Síntese em Fase Sólida / Afinidade de Anticorpos Idioma: En Revista: Nucleic Acids Res Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Suécia

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Uracila / Sítios de Ligação de Anticorpos / Engenharia de Proteínas / Mutagênese Sítio-Dirigida / Técnicas de Síntese em Fase Sólida / Afinidade de Anticorpos Idioma: En Revista: Nucleic Acids Res Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Suécia