Abstract
The GlnAP2 element has been proved to be an effective and inducible—by exogenous acetate—promoter in Escherichia coli with glnL/pta double mutations. Based on this feature, a single-copy expression vector was constructed via coupling of the glnAP2 promoter-regulated T7 RNA polymerase gene and the T7-promoter-controlled lacZ gene on a bacterial artificial chromosome. After induction with 20 mM potassium acetate, the glnL/pta double mutant E. coli harboring the single-copy plasmid produced 47,500 Miller units of β-galactosidase activity. This high level expression, corresponding to 27% of total cell protein, was comparable to that determined with the commercial multi-copy expression vector, pET-14b, in strain E. coli Tuner (DE3) (64,300 Miller units, 41% of total cell protein). Moreover, this single-copy expression vector could be maintained for at least 150 generations even in the presence of inducers. In contrast, the multi-copy expression vector was extensively lost after induction. The results indicate that the single-copy expression system has the potential for high-level heterologous protein production for industrial applications.
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Acknowledgement
We are grateful to Professor James Liao, University of California, Los Angeles, USA for providing us with E. coli BW18793 and for many valuable discussions.
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Chang, TS., Wu, WJ., Wan, HM. et al. High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli . Appl Microbiol Biotechnol 61, 234–239 (2003). https://doi.org/10.1007/s00253-003-1252-4
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DOI: https://doi.org/10.1007/s00253-003-1252-4