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The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli
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 subsequen...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Medknow Publications & Media Pvt Ltd
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698866/ https://www.ncbi.nlm.nih.gov/pubmed/26779275 |
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author | Haddad, Leila Babaeipour, Valiollah Mofid, Mohammad Reza |
author_facet | Haddad, Leila Babaeipour, Valiollah Mofid, Mohammad Reza |
author_sort | Haddad, Leila |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4698866 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Medknow Publications & Media Pvt Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-46988662016-01-15 The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli Haddad, Leila Babaeipour, Valiollah Mofid, Mohammad Reza Res Pharm Sci Original Article 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. Medknow Publications & Media Pvt Ltd 2015 /pmc/articles/PMC4698866/ /pubmed/26779275 Text en Copyright: © Research in Pharmaceutical Sciences http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. |
spellingShingle | Original Article Haddad, Leila Babaeipour, Valiollah Mofid, Mohammad Reza The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title | The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title_full | The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title_fullStr | The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title_full_unstemmed | The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title_short | The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli |
title_sort | effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in e. coli |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698866/ https://www.ncbi.nlm.nih.gov/pubmed/26779275 |
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