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 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).
Integrated Biotechnology Laboratory, School of Life Sciences, Anhui University, Hefei, 230601, China.
Zhao-Yuan
ZY
Chen
Jie
J
Cao
Li
L
Xie
Xiao-Fei
XF
Li
Zhen-Hai
ZH
Yu
Wang-Yu
WY
Tong
D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D017353:Gene Deletion; D042822:Genomic Instability; D060847:Metabolic Engineering; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D013879:Thioredoxins; C087896:reteplase; D010959:Tissue Plasminogen Activator