Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies

Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized process...

Descripción completa

Detalles Bibliográficos
Autores principales: Esmaili, Iman, Mohammad Sadeghi, Hamid Mir, Akbari, Vajihe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Medknow Publications & Media Pvt Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082031/
https://www.ncbi.nlm.nih.gov/pubmed/30271443
http://dx.doi.org/10.4103/1735-5362.236834
_version_ 1783345750203170816
author Esmaili, Iman
Mohammad Sadeghi, Hamid Mir
Akbari, Vajihe
author_facet Esmaili, Iman
Mohammad Sadeghi, Hamid Mir
Akbari, Vajihe
author_sort Esmaili, Iman
collection PubMed
description Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding.
format Online
Article
Text
id pubmed-6082031
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Medknow Publications & Media Pvt Ltd
record_format MEDLINE/PubMed
spelling pubmed-60820312018-10-01 Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies Esmaili, Iman Mohammad Sadeghi, Hamid Mir Akbari, Vajihe Res Pharm Sci Original Article Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding. Medknow Publications & Media Pvt Ltd 2018-10 /pmc/articles/PMC6082031/ /pubmed/30271443 http://dx.doi.org/10.4103/1735-5362.236834 Text en Copyright: © 2018 Research in Pharmaceutical Sciences http://creativecommons.org/licenses/by-nc-sa/4.0 This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
spellingShingle Original Article
Esmaili, Iman
Mohammad Sadeghi, Hamid Mir
Akbari, Vajihe
Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title_full Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title_fullStr Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title_full_unstemmed Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title_short Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
title_sort effect of buffer additives on solubilization and refolding of reteplase inclusion bodies
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082031/
https://www.ncbi.nlm.nih.gov/pubmed/30271443
http://dx.doi.org/10.4103/1735-5362.236834
work_keys_str_mv AT esmailiiman effectofbufferadditivesonsolubilizationandrefoldingofreteplaseinclusionbodies
AT mohammadsadeghihamidmir effectofbufferadditivesonsolubilizationandrefoldingofreteplaseinclusionbodies
AT akbarivajihe effectofbufferadditivesonsolubilizationandrefoldingofreteplaseinclusionbodies