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Auto-induction expression of human consensus interferon-alpha in Escherichia coli

BACKGROUND: Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or va...

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Autores principales: EL-Baky, Nawal Abd, Linjawi, Mustafa H, Redwan, Elrashdy M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372041/
https://www.ncbi.nlm.nih.gov/pubmed/25886839
http://dx.doi.org/10.1186/s12896-015-0128-x
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author EL-Baky, Nawal Abd
Linjawi, Mustafa H
Redwan, Elrashdy M
author_facet EL-Baky, Nawal Abd
Linjawi, Mustafa H
Redwan, Elrashdy M
author_sort EL-Baky, Nawal Abd
collection PubMed
description BACKGROUND: Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a. RESULTS: The expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a. CONCLUSIONS: The auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production.
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spelling pubmed-43720412015-03-25 Auto-induction expression of human consensus interferon-alpha in Escherichia coli EL-Baky, Nawal Abd Linjawi, Mustafa H Redwan, Elrashdy M BMC Biotechnol Research Article BACKGROUND: Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a. RESULTS: The expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a. CONCLUSIONS: The auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production. BioMed Central 2015-03-06 /pmc/articles/PMC4372041/ /pubmed/25886839 http://dx.doi.org/10.1186/s12896-015-0128-x Text en © EL-Baky et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
EL-Baky, Nawal Abd
Linjawi, Mustafa H
Redwan, Elrashdy M
Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title_full Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title_fullStr Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title_full_unstemmed Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title_short Auto-induction expression of human consensus interferon-alpha in Escherichia coli
title_sort auto-induction expression of human consensus interferon-alpha in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372041/
https://www.ncbi.nlm.nih.gov/pubmed/25886839
http://dx.doi.org/10.1186/s12896-015-0128-x
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