title: A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host.
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.
authors.affiliation: 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. lyngsoe@sund.ku.dk
authors.forename: Christina
authors.initials: C
authors.lastname: Lyngsø
authors.forename: Søren
authors.initials: S
authors.lastname: Kjaerulff
authors.forename: Sven
authors.initials: S
authors.lastname: Müller
authors.forename: Tomas
authors.initials: T
authors.lastname: Bratt
authors.forename: Uffe H
authors.initials: UH
authors.lastname: Mortensen
authors.forename: Florence
authors.initials: F
authors.lastname: Dal Degan
mesh_terms: D005816:Genetic Complementation Test; D006801:Humans; D019151:Peptide Library; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D012568:Schizosaccharomyces / Q000235:genetics; D012641:Selection, Genetic; D020539:Sequence Analysis, Protein / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics
chemical_list: D019151:Peptide Library; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha