High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli.
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.
Chang
T-S
TS
Department of Chemical Engineering, National Tsing Hua University, 30043, Hsinchu, Taiwan. wtwu@che.nthu.edu.tw
Wu
W-J
WJ
Wan
H-M
HM
Shiu
T-R
TR
Wu
W-T
WT
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007763:Lac Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001616:beta-Galactosidase