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

TY - JOUR

T1 - A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host.

AU - Lyngsø, Christina

AU - Kjaerulff, Søren

AU - Muller, Sven

AU - Bratt, Tomas

AU - Mortensen, Uffe H.

AU - Dal Degan, Florence

N1 - Paper id:: 20082307

PY - 2010/3

Y1 - 2010/3

N2 - 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. Published by Wiley-Blackwell.

AB - 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. Published by Wiley-Blackwell.

U2 - 10.1002/pro.337

DO - 10.1002/pro.337

M3 - Journal article

C2 - 20082307

SN - 0961-8368

VL - 19

SP - 579

EP - 592

JO - Protein Science

JF - Protein Science

IS - 3

ER -