RECOMBINANT PROTEIN-PRODUCTION IN CULTURES OF AN ESCHERICHIA-COLI TRP- STRAIN

Fermentation conditions were developed in order to achieve simultaneou sly a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is u nder control of the Escherichia coli trp promoter, in a trp derivative strain of E. coli W31 10. The dual role of tryptophan concentration o n cellular growth and hybrid gene regulation was studied in 10-1 batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hyb rid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cel ls retained the recombinant plasmid. In order to increase the hybrid p rotein production level, a fed-batch culture strategy was developed wh ereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possibl e to increase the final biomass concentration to 20 g/l, plasmid-beari ng cells in the population to 90% and recombinant hybrid protein to 1. 21 g/l.