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Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli
Authors:Zhao‐Yuan Chen  Jie Cao  Li Xie  Xiao‐Fei Li  Zhen‐Hai Yu  Wang‐Yu Tong
Institution:Integrated Biotechnology Laboratory, School of Life Sciences, Anhui University, , Hefei, 230601 China
Abstract: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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