PMID:12698281
| Citation |
Chang, TS, Wu, WJ, Wan, HM, Shiu, TR and Wu, WT (2003) High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli. Appl. Microbiol. Biotechnol. 61:234-9 |
|---|---|
| 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 beta-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. |
| Links |
PubMed Online version:10.1007/s00253-003-1252-4 |
| Keywords |
Chromosomes, Artificial, Bacterial/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Gene Expression; Lac Operon/genetics; Plasmids/genetics; Promoter Regions, Genetic/genetics; Transcription, Genetic/genetics; beta-Galactosidase/analysis; beta-Galactosidase/genetics |
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