期刊
FEBS JOURNAL
卷 279, 期 17, 页码 3176-3184出版社
WILEY
DOI: 10.1111/j.1742-4658.2012.08697.x
关键词
batch-mode cell-free protein expression; disulfide bond formation; Escherichia coli S30 cell extract; NMR spectroscopy; stable-isotope labeling
资金
- ETH Zurich
- Swiss National Science Foundation through National Center of Competence in Research (NCCR) Structural Biology
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 0.30.7 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.
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