Fermentation conditions for high‐level expression of the tac‐promoter‐controlled calf prochymosin cDNA in Escherichia coli HB101

Abstract

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate

“…Expression of recombinant proteins generally inhibits the specific growth rate of E. coli (Lee et al, 1988;Jensen and Carlsen, 1990;Kaprálek et al, 1991;Olsson et al, 1996;Panda et al, 1999) but the degree of inhibition depends on the protein expressed. Automatic inducer addition and harvesting based on on-line estimates of biomass concentration are particularly useful in cases where the degree of inhibition of the specific growth rate is unknown since the system allows the induction period to be controlled relative to changes of biomass concentration.…”

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

“…Although several studies have been performed in order to optimize the induction of gene expression by using lactose (Donovan et al, 1996;Gombert and Kilikian, 1998;Kapralek et al, 1991;Kilikian et al, 2000;Neubauer et al, 1991;Neubauer and Hofmann, 1994;Woyski and CuppVickery, 2001), surprisingly none of them addressed the problem of the Lac nongenetic population heterogeneity (Chanal et al, 1998;Maloney and Rotman, 1973;Novick and Weiner, 1957;Tolker-Nielsen et al, 1998), nor the gal − genotype present in the most commonly used strain for gene expression, BL21. In order to overcome these limitations and to obtain an "ideal" strain for the expression of heterologous proteins in E. coli, using lactose as inducer, we introduced two genetic modifications into BL21 to obtain MP101, a lacO c gal + strain.…”

“…In Lac + host strains the promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source (Donovan et al, 1996;Gombert and Kilikian, 1998;Kapralek et al, 1991;Kilikian et al, 2000;Neubauer et al, 1991;Neubauer and Hofmann, 1994;Woyski and Cupp-Vickery, 2001). Despite its low cost, the use of lactose as inducer has several drawbacks, which results in low volumetric yields and difficulties in process control (Gombert and Kilikian, 1998).…”

Production of Active Mammalian and Viral Proteases in Bacterial Expression Systems

“…EventuaHy, by using the tae pronloter, enzymatically active chymosin could be produced at 12 mg per litre of culture broth (Kapralek et al, 1990)~The expression of chymosin has also been used as a nl0deI systeJn in the development of more efficient ribosomal binding sites (Emtage et al, 1983;Kawaguchi et al, 1986;Wang et a/., 1995a). In addition, active chymosin has been produced in the culture Inediulll by secretion from protoplast type L-forms of Proteus I1zirabilis at 17 lng/litre by using the streptococcal pyrogenic exotoxin type A pronloter and signal sequence fused to the pro-chymosin gene (Klessen et aI., 1989).…”