title: Cell-free expression of disulfide-containing eukaryotic proteins for structural biology.
abstract: We describe Escherichia coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3-0.7 mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy.
authors: Institute of Molecular Biology and Biophysics, ETH Zurich, Switzerland.
authors: Erich
authors: E
authors: Michel
authors: Kurt
authors: K
authors: Wüthrich
mesh_terms: D000818:Animals; D002384:Catalysis; D002474:Cell-Free System; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D004220:Disulfides; D011506:Proteins