title: High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli.
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.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 30043, Hsinchu, Taiwan. wtwu@che.nthu.edu.tw
authors.forename: T-S
authors.initials: TS
authors.lastname: Chang
authors.forename: W-J
authors.initials: WJ
authors.lastname: Wu
authors.forename: H-M
authors.initials: HM
authors.lastname: Wan
authors.forename: T-R
authors.initials: TR
authors.lastname: Shiu
authors.forename: W-T
authors.initials: WT
authors.lastname: Wu
mesh_terms: 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
chemical_list: D001616:beta-Galactosidase