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Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein

Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of diff...

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Autores principales: Rasooli, Farideh, Hashemi, Atieh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wolters Kluwer - Medknow 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937747/
https://www.ncbi.nlm.nih.gov/pubmed/32038735
http://dx.doi.org/10.4103/1735-5362.272564
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author Rasooli, Farideh
Hashemi, Atieh
author_facet Rasooli, Farideh
Hashemi, Atieh
author_sort Rasooli, Farideh
collection PubMed
description Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated. Moreover, the effect of temperature and isopropyl-β-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), Rosetta™ (DE3), and Origami™ (DE3). The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in Rosetta™ (DE3) and Origami™ (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.
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spelling pubmed-69377472020-02-07 Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein Rasooli, Farideh Hashemi, Atieh Res Pharm Sci Original Article Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated. Moreover, the effect of temperature and isopropyl-β-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), Rosetta™ (DE3), and Origami™ (DE3). The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in Rosetta™ (DE3) and Origami™ (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system. Wolters Kluwer - Medknow 2019-12-11 /pmc/articles/PMC6937747/ /pubmed/32038735 http://dx.doi.org/10.4103/1735-5362.272564 Text en Copyright: © 2019 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
Rasooli, Farideh
Hashemi, Atieh
Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title_full Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title_fullStr Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title_full_unstemmed Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title_short Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein
title_sort efficient expression of epex in the cytoplasm of escherichia coli using thioredoxin fusion protein
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937747/
https://www.ncbi.nlm.nih.gov/pubmed/32038735
http://dx.doi.org/10.4103/1735-5362.272564
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