High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli.
Appl Microbiol Biotechnol
; 61(3): 234-9, 2003 May.
Article
em En
| MEDLINE
| ID: mdl-12698281
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.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Expressão Gênica
/
Cromossomos Artificiais Bacterianos
/
Escherichia coli
/
Óperon Lac
Idioma:
En
Ano de publicação:
2003
Tipo de documento:
Article