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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...

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Autores principales: Chen, Zhao-Yuan, Cao, Jie, Xie, Li, Li, Xiao-Fei, Yu, Zhen-Hai, Tong, Wang-Yu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BlackWell Publishing Ltd 2014
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).
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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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