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Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli
In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinan...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BlackWell Publishing Ltd
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241728/ https://www.ncbi.nlm.nih.gov/pubmed/24779863 http://dx.doi.org/10.1111/1751-7915.12127 |
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author | Chen, Zhao-Yuan Cao, Jie Xie, Li Li, Xiao-Fei Yu, Zhen-Hai Tong, Wang-Yu |
author_facet | Chen, Zhao-Yuan Cao, Jie Xie, Li Li, Xiao-Fei Yu, Zhen-Hai Tong, Wang-Yu |
author_sort | Chen, Zhao-Yuan |
collection | PubMed |
description | In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(−1). |
format | Online Article Text |
id | pubmed-4241728 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BlackWell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-42417282014-12-08 Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli Chen, Zhao-Yuan Cao, Jie Xie, Li Li, Xiao-Fei Yu, Zhen-Hai Tong, Wang-Yu Microb Biotechnol Research Articles In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(−1). BlackWell Publishing Ltd 2014-07 2014-04-30 /pmc/articles/PMC4241728/ /pubmed/24779863 http://dx.doi.org/10.1111/1751-7915.12127 Text en © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Chen, Zhao-Yuan Cao, Jie Xie, Li Li, Xiao-Fei Yu, Zhen-Hai Tong, Wang-Yu Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title | Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title_full | Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title_fullStr | Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title_full_unstemmed | Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title_short | Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli |
title_sort | construction of leaky strains and extracellular production of exogenous proteins in recombinant escherichia coli |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241728/ https://www.ncbi.nlm.nih.gov/pubmed/24779863 http://dx.doi.org/10.1111/1751-7915.12127 |
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