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Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology

Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Escherichia coli leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, con...

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Autores principales: Ahmadian, Mina, Jahanian-Najafabadi, Ali, Akbari, Vajihe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Shaheed Beheshti University of Medical Sciences 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758011/
https://www.ncbi.nlm.nih.gov/pubmed/33680031
http://dx.doi.org/10.22037/ijpr.2020.1101169
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author Ahmadian, Mina
Jahanian-Najafabadi, Ali
Akbari, Vajihe
author_facet Ahmadian, Mina
Jahanian-Najafabadi, Ali
Akbari, Vajihe
author_sort Ahmadian, Mina
collection PubMed
description Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Escherichia coli leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, concentrations and pHs of denaturing agents and addition of reducing agents on the yield of inclusion bodies solubilization was evaluated. Next, various conditions were evaluated for refolding hGM-CSF using a two-step design of experiment (DOE) including primary screening by factorial design, and then optimization by response surface design. It was found that hGM-CSF inclusion bodies can be efficiently solubilized with 4 M urea and 4 mM β-mercaptoethanol, pH = 9. A response surface quadratic model was employed to predict the optimum refolding conditions and the accuracy of this model was confirmed by high value of R(2) (0.99) and F-value of 0.64. DOE results revealed that sorbitol (0.235 M), imidazole (97 mM), and SDS (0.09%) would be the optimum buffer additives for refolding of hGM-CSF. Following refolding studies, the obtained protein was subjected to circular dichroism which confirmed correct secondary structure of the refolded hGM-CSF. The refolded hGM-CSF exhibited reasonable biological activity compared with standard protein. The approach developed in this work can be important to improve the refolding of other proteins with similar structural features.
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spelling pubmed-77580112021-03-05 Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology Ahmadian, Mina Jahanian-Najafabadi, Ali Akbari, Vajihe Iran J Pharm Res Original Article Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Escherichia coli leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, concentrations and pHs of denaturing agents and addition of reducing agents on the yield of inclusion bodies solubilization was evaluated. Next, various conditions were evaluated for refolding hGM-CSF using a two-step design of experiment (DOE) including primary screening by factorial design, and then optimization by response surface design. It was found that hGM-CSF inclusion bodies can be efficiently solubilized with 4 M urea and 4 mM β-mercaptoethanol, pH = 9. A response surface quadratic model was employed to predict the optimum refolding conditions and the accuracy of this model was confirmed by high value of R(2) (0.99) and F-value of 0.64. DOE results revealed that sorbitol (0.235 M), imidazole (97 mM), and SDS (0.09%) would be the optimum buffer additives for refolding of hGM-CSF. Following refolding studies, the obtained protein was subjected to circular dichroism which confirmed correct secondary structure of the refolded hGM-CSF. The refolded hGM-CSF exhibited reasonable biological activity compared with standard protein. The approach developed in this work can be important to improve the refolding of other proteins with similar structural features. Shaheed Beheshti University of Medical Sciences 2020 /pmc/articles/PMC7758011/ /pubmed/33680031 http://dx.doi.org/10.22037/ijpr.2020.1101169 Text en This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Ahmadian, Mina
Jahanian-Najafabadi, Ali
Akbari, Vajihe
Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title_full Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title_fullStr Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title_full_unstemmed Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title_short Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology
title_sort optimization of buffer additives for efficient recovery of hgm-csf from inclusion bodies using response surface methodology
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758011/
https://www.ncbi.nlm.nih.gov/pubmed/33680031
http://dx.doi.org/10.22037/ijpr.2020.1101169
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