Dynamics of induced CAT expression in E. coli

Abstract

The dynamics of chemically induced chloramphenicolaceyl-transferase (CAT) expression are determined in batch cultures of Escherichia coli DH5alphaF'IQ [pKK262-1]. This article is directed towards understanding the coupling of induced cloned-protein synthesis and reduced cell growth which are balanced in the optimal system. Experimental results indicate that the best inducer (IPTG) concentration is near 1.0 mM when added during midexponential growth. Lower concentrations cause only weak induction, whereas higher concentrations cause sufficiently strong induction that cell growth is suppressed. Induction at the onset of the stationary phase results in high expression but is accompanied by stimulated protease activity. Also, cell mass yield is adversely affected by enhanced protein synthesis. A structured metabolic model is shown to predict the responses of instantaneous growth rate and productivity which result from protein overexpression. This model can be employed to predict alternative reactor strategies and operating conditions necessary for the design of efficient bioprocess.

Recombinant protein production in an Escherichia coli reduced genome strain

“…As expected, the high induction strength resulted in higher specific CAT activity (Figure 2) for both the low and high fed-batch growth rate fermentations, although not proportionally higher. A nonproportional increase in recombinant protein expression with respect to induction strength has been observed in other E coli strains (Bentley et al, 1991;Ramirez and Bentley, 1995).…”

Parameters Affecting Gene Expression from the Pm Promoter in Gram-Negative Bacteria

“…In general it is well known that high gene expression levels in combination with high copy number plasmids can lead to reduced cell growth and viability (Bentley et al, 1991;Betenbaugh et al, 1989). In addition, there seems to be more specific problems associated with high copy number RK2 replicons (Haugan et al, 1995).…”

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

“…Unfortunately, this equation is inappropriate when foreign protein is expressed. The metabolic burden placed on the cell during recombinant protein production has been well documented (Bentley et al, 1990;Glick, 1995) and is observed macroscopically as a reduction in growth rate (Bentley et al, 1991;Zabriskie et al, 1986). To model the mitigating effect that foreign protein expression has on the specific growth rate, the Monod expression is multiplied by a product inhibition term as follows:…”