Coupling the T7 A1 promoter to the runaway-replication vector as an efficient method for stringent control and...

An expression vector characterized by tight regulation and high expression of cloned genes appears to be indispensable for the engineering need. To achieve this goal, in association with lacI the T7 A1 promoter containing two synthetic lac operators was constructed into a runaway-replication vector. To further examine this vector system, lacZ was subcloned and placed under the control of the T7 A1 promoter on the plasmid. With the application of the thermal induction alone, the Escherichia coli strain harboring the recombinant plasmid was able to produce 15,000 Miller units of beta-galactosidase, while it yielded the recombinant protein with 45,000-50,000 Miller units upon both thermal and chemical induction. In sharp contrast, only 60-90 Miller units of beta-galactosidase was obtained for the cell at an uninduced state. As a result, the production yield of beta-galactosidase over the background level is amplified approximately 170-fold by thermal induction and 500-fold by thermal and chemical induction. To produce the recombinant protein on a large scale, an approach by connecting two fermenters in series was newly developed. By applying the three-stage temperature shift in this dual fermenter system, 55,000 Miller units of beta-galactosidase was obtained. Overall, it shows the potential use of the vector system developed here for its tight control and high production of recombinant proteins.