A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

C. Lyngsø, S. Kjaerulff, S. Muller, T. Bratt, Uffe Hasbro Mortensen, F. Dal Degan

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality-control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF-alpha, from which different folding competent mutant proteins were uncovered.
    Original languageEnglish
    JournalProtein Science
    Volume19
    Issue number3
    Pages (from-to)579-592
    ISSN0961-8368
    DOIs
    Publication statusPublished - 2010

    Keywords

    • protein engineering
    • protein folding
    • heterologous expression
    • fission yeast
    • eukaryote
    • in vivo selection
    • TNF-alpha
    • folding quality control
    • solubility
    • selection
    • recombinant protein
    • library construction
    • yeast
    • protein vaccine
    • Schizosaccharomyces pombe

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