Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions

Eeva-Christine Brockmann (Corresponding Author), Matthew Cooper, Nelli Strömsten, Markus Vehniäinen, Petri Saviranta

    Research output: Contribution to journalArticleScientificpeer-review

    48 Citations (Scopus)

    Abstract

    Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.
    Original languageEnglish
    Pages (from-to)159 - 170
    Number of pages12
    JournalJournal of Immunological Methods
    Volume296
    Issue number1-2
    DOIs
    Publication statusPublished - 2005
    MoE publication typeA1 Journal article-refereed

    Keywords

    • Antibody engineering
    • Disulfide bond
    • Phage display
    • Protein denaturation
    • Protein stability

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