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A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host
Author(s) -
Lyngsø Christina,
Kjaerulff Søren,
Müller Sven,
Bratt Tomas,
Mortensen Uffe H.,
Dal Degan Florence
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.337
Subject(s) - complementation , protein folding , fusion protein , biology , schizosaccharomyces pombe , microbiology and biotechnology , directed evolution , folding (dsp implementation) , schizosaccharomyces , mutant , protein engineering , biochemistry , gene , recombinant dna , enzyme , engineering , electrical engineering
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.