Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: I. Batch cultures and kinetic modeling

Abstract

A novel Eschericha coli expression system directed by bacteriophage T7 RNA Polymerase utilized for overexpression of the cloned gene. The recombinant cell contains the plasmid with a bacteriophage promoter, the T7 promoter, to regulate the expression of the target gene. This promoter is recognized only by T7 RNA polymerase, whose gene has been fused into the host chromosome and is under control of the lacUV5 promoter. Therefore, the target gene on the plasmid can be expressed only in the presence of T7 RNA polymerase, which is induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The batch cultures were performed to investigate the effect of induction on kinetics of cell growth and foreign protein formation and to determine the optimal induction strategy. It was observed that the specific growth rates of the recombinant cells dramatically decrease after induction, and that there is an optimal induction time for maximizing the accumulated intracellular foreign protein. This optimal induction time varies significantly with inducer concentration. To better understand the optimal behavior, a lumped mechanistic model was constructed to analyze the induced cell growth and foreign protein formation rates.

High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

“…Generally, it is well established that IPTG induction can decrease cell growth. 18,19 However, the opposite effect was observed in our assays with M15. A variation of the correlation g DCW/OD 600 nm was identified as an explanation for this incongruence.…”

Model-based optimization of viral capsid protein production in fed-batch culture of recombinant Escherichia coli

“…This showed that our measurement data was accurate when compared with the representation proposed here. However, with respect to process optimization, the approach [13] requires additional state variables and more complex kinetics to avoid unrealistic control profiles. The latter tend to maintain a specific biomass growth rate in the protein production phase close to zero.…”

“…This approach can be considered as a form of self-inhibition effect [13]. However, we assume that the maximal specific product concentration value p max , approached asymptotically, to be dependent on l. p max was observed to decrease with higher biomass growth rates l, but is expected to be very low when the specific biomass growth rate l approaches zero.…”

“…The conventional approach used for example by Miao and Kompala [13] and Bellgardt [14] was tested for its ability to describe the measurement data presented in this paper. This showed that our measurement data was accurate when compared with the representation proposed here.…”

Framework for online optimization of recombinant protein expression in high-cell-densityEscherichia coli cultures using GFP-fusion monitoring

“…However, there have been few reports of foreign production models that consider induction effects (Bentley et al, 1991;Betenbaugh and Dhurjati, 1990;Lee and Ramirez, 1992;Miao and Kompala, 1992). Lee and Ramirez (1992) proposed a mathematical model that included inducer effects on cell growth and foreign protein production.…”