Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lac UV5par8EGF, both encoding human epidermal growth factor (hEGF), were used i n fermentations to optimize levels of excreted hEGF. Medium composition, in ducer level, growth stage at induction and culture conditions, were optimiz ed with respect to volumetric production of the recombinant protein. MMBL m edium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. I sopropyl-beta-D-thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were f ound to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continu ous fed-batch process for cultivation of E. call JM101[pWKW2] was developed . The maximum concentration of excreted hEGF attained in continuous fed-bat ch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantia ted by SDS-PAGE and immunoblotting. (C) 1999 Elsevier Science Ltd. All righ ts reserved.