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KMID : 0380020060210020118
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Korean Journal of Biotechnology and Bioengineering
2006 Volume.21 No. 2 p.118 ~ p.122
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Enhanced Synthesis of Active rPA in the Continuous Exchange Cell-free Protein Synthesis (CECF) System utilizing Molecular Chaperones
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Park Chang-Gil
Kim Tae-Wan
Choi Cha-Yong
Kim Dong-Myung
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Abstract
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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 ㎍/㎖ 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.
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KEYWORD
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rPA, CECF, continuous exchange cell-free protein synthesis, molecular chaperone, IAM
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