The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli

L Haddad, V Babaeipour, MR Mofid

Abstract


The isolation of the target protein from inclusion bodies (IBs) is a preliminary step to increase protein titer and to maintain its biological activity. In the present study, the effects of various cell lysis methods and the expression temperature was investigated on the improvement of the subsequent purification steps of mecasermin produced in IB. We also investigated the solubilization profile of the top-notch IB in 6 M guanidine hydrochloride (Gdn-HCl) and 8 M urea at different pH ranges. Mecasermin was expressed at various temperatures (25, 28, 30, and 37 °C) and the Escherichia coli cells were lysed by different cell lysis methods. The purity and quality of harvested IBs was evaluated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, mecasermin was refolded and purified using gel filtration chromatography. The profile of SDS-PAGE analysis showed higher quality and purity after application of sonication coupled with lysozyme pretreatment for expressed mecasermin at 37 °C. Besides, from dithiothreitol application in washing step, we achieved a manifold enriched secondary IB for further purification of mecasermin. Mecasermin exhibited optimized solubility in 6 M Gdn-HCl at pH of 5.4. The findings of this study indicate an important role for cell disruption techniques to efficient purification of mecasermin. The study presents the most efficient techniques for improvement of downstream purification of mecasermin.


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