OVERPRODUCTION OF A RECOMBINANT CARBOXYPEPTIDASE INHIBITOR BY OPTIMIZATION OF FERMENTATION CONDITIONS

In order to optimize the production of recombinant potato carboxypepti dase inhibitor (rePCI), a protein with 39 amino acid residues and thre e disulphide bridges, by Escherichia coli MC1061[pIMAM3], the effects of various parameters were investigated. Production of rePCI in M9CAS medium was optimal at 37 degrees C and using low concentrations of gly cerol as a carbon source. Increasing concentrations of glycerol caused a decrease in the production of rePCI, which was almost totally inhib ited above 1% glycerol. Relatively high concentrations of oligoelement s in the culture medium also inhibited the production of rePCI. We obt ained a 100-fold increase in the production of rePCI, from 2 to 200 mg /l, when growing bacteria in a fed-batch aerobic culture using a 2-1 f ermenter. RePCI was found exclusively in the supernatant, although the genetic construction was designed for it to be released into the peri plasmic space. Large quantities of rePCI could be easily purified from the supernatants of these cultures. Our conditions of fed-batch, aero bic fermentation could be used for overproduction to high levels of ot her recombinant proteins.