Mass production of human epidermal growth factor using fed‐batch cultures of recombinant Escherichia coli

Abstract

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.

Proposed mechanism of acetate accumulation in two recombinantEscherichia coli strains during high density fermentation

“…Glucose is the preferred carbon source for these fermentations; it is being used for energy production and for the synthesis of various metabolites through the glycolysis and tricarboxylic acid (TCA) pathways (Holms, 1986;Nimmo, 1987;Nunn, 1987). Acetic acid is a by-product of the glucose metabolism, and its level can affect the productivity of the fermentation process by slowing the bacterial growth and/or inhibiting recombinant-protein biosynthesis (Bentley et al, 1990;Han et al, 1992;Ko et al, 1995;Shimizu et al, 1991).…”

Applications, and Efficient Large-Scale Production, of Recombinant Human Epidermal Growth Factor

“…Several studies prior to 1993 addressed the molecular biology of recombinant strain construction, but EGF yields were low (in the Ilg/ml range). As examples of this pioneering work, Kim et al (1992) described low-level production of EGF by Escherichia coli in continuous culture, while Shimizu et al (1991) developed a fed-batch procedure for recombinant EOF production.…”

Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer

“…However, the effect of postinduction nutrient feeding strategies has received less attention. Due to the detrimental effect of acetate on recombinant protein production (Bech Jensen and Carlsen, 1990;MacDonald and Neway, 1990;Shimizu et al, 1992), its formation during postinduction feeding caused by carbon overflux must be prevented. At this stage, specific growth rate (and acetate production) cannot be maintained at a constant value by exponential feeding of nutrients, due to the changes in cell physiology produced by the expression of the recombinant protein (Wong et al, 1998).…”