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Enhanced Synthesis of Active rPA in the Continuous Exchange Cell-free Protein Synthesis [CECF] System utilizing Molecular Chaperones  

Park, Chang-Gil (Department of Fine Chemical Engineering and Chemistry, School of Engineering, Chungnam National University)
Kim, Tae-Wan (School of Chemical and Biological Engineering, College of Engineering, Seoul National University)
Choi, Cha-Yong (School of Chemical and Biological Engineering, College of Engineering, Seoul National University)
Kim, Dong-Myung (Department of Fine Chemical Engineering and Chemistry, School of Engineering, Chungnam National University)
Publication Information
KSBB Journal / v.21, no.2, 2006 , pp. 118-122 More about this Journal
Abstract
In this report, we describe that the use of GroEL/GroES-enriched S30 extract remarkably enhances the solubility and enzymatic activity of cell-free synthesized rPA, which requires the correct formation of 9 disulfide bonds for its biological activity. We found that the stable maintenance of redox potential is necessary, but not sufficient for the optimal expression of active rPA. In a control reaction without using additional molecular chaperones, most of the rPA molecules were aggregated almost instantly after their expression and thus failed to exhibit the enzymatic activity. However, by the use of GroEL/GroES-enriched extract, combined with IAM-treatment, approximately $30{\mu}g/ml$ of active rPA was expressed in the cell-free synthesis reaction. This result not only demonstrates the efficient production of complex proteins, but also shows the control and flexibility offered by the cell-free protein synthesis system.
Keywords
rPA; CECF(continuous exchange cell-free protein synthesis); molecular chaperone; IAM;
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