A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host |
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Authors: | Christina Lyngsø Søren Kjaerulff Sven Müller Tomas Bratt Uffe H Mortensen Florence Dal Degan |
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Institution: | 1. The Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3B, DK‐2200 Copenhagen N, Denmark;2. Pharmexa A/S, Agern Allé 1, DK‐2970 Hoersholm, Denmark;3. Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, DK‐2800 Lyngby, Denmark;4. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, DK‐2200 Copenhagen N, Denmark |
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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‐α, from which different folding competent mutant proteins were uncovered. |
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Keywords: | protein folding selection protein engineering folding quality control heterologous expression eukaryote in vivo selection library construction solubility TNF‐α recombinant protein Schizosaccharomycespombe yeast fission yeast protein vaccine |
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