Maximizing the expression of recombinant proteins in Escheria coli by manipulation of culture conditions

The expression of hybrid proteins β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.