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Production of recombinant protein by a novel oxygen-induced system in Escherichia coli
BACKGROUND: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared wit...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234026/ https://www.ncbi.nlm.nih.gov/pubmed/24708849 http://dx.doi.org/10.1186/1475-2859-13-50 |
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author | Baez, Antonino Majdalani, Nadim Shiloach, Joseph |
author_facet | Baez, Antonino Majdalani, Nadim Shiloach, Joseph |
author_sort | Baez, Antonino |
collection | PubMed |
description | BACKGROUND: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. RESULTS: The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). CONCLUSIONS: Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery. |
format | Online Article Text |
id | pubmed-4234026 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-42340262014-11-18 Production of recombinant protein by a novel oxygen-induced system in Escherichia coli Baez, Antonino Majdalani, Nadim Shiloach, Joseph Microb Cell Fact Research BACKGROUND: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. RESULTS: The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). CONCLUSIONS: Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery. BioMed Central 2014-04-07 /pmc/articles/PMC4234026/ /pubmed/24708849 http://dx.doi.org/10.1186/1475-2859-13-50 Text en Copyright © 2014 Baez et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 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 Baez, Antonino Majdalani, Nadim Shiloach, Joseph Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title | Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title_full | Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title_fullStr | Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title_full_unstemmed | Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title_short | Production of recombinant protein by a novel oxygen-induced system in Escherichia coli |
title_sort | production of recombinant protein by a novel oxygen-induced system in escherichia coli |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234026/ https://www.ncbi.nlm.nih.gov/pubmed/24708849 http://dx.doi.org/10.1186/1475-2859-13-50 |
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