Synthesis of recombinant protein (human mini-proinsulin) is investigated in fed-batch cultures at high cell concentration of recombinant Escherichia coli BL21(DE3)[pET-3aT2M2]. Transcription of the recombinant gene is controlled by a T7 promoter system. The human mini-proinsulin is characterized by a C-chain peptide consisting of only nine amino acids, whereas the C-chain peptide of natural human proinsulin is made up of 35 amino acids. It is expressed in a fusion protein with a small fusion partner (a peptide with 18 amino acids) and finally aggregated into insoluble inclusion bodies in cytoplasm of recombinant E. coli. The fermentative production of this small fusion mini-proinsulin may be of great advantage in enhancing the yield of human insulin. To find an optimum induction strategy, effects of various key cultivation variables on the mini-proinsulin production are examined in high cell density fed-batch cultures. No general correlation is found between preinduction specific growth rate and recombinant protein synthesis, which confers a flexibility in choosing the feeding strategy of preinduction media for achieving the high cell density cultures. A culture temperature below 37 degrees C is unfavorable for recombinant gene expression, and the T7-based expression system is almost completely repressed at 30 degrees C. The nutrient glucose and yeast extract concentration in postinduction feed media is optimized by applying a statistical method for medium optimization, i.e. response surface methodology, and an effective amount of inducer molecule (IPTG) is determined to maximize the specific recombinant protein formation. The mini-proinsulin production in E. coli culture is significantly influenced by the volumetric feed rate of postinduction media, which is shown to be closely related to the plasmid copy number in the recombinant cell. Consequently, in a single-stage fed-batch process, the mini-proinsulin concentration is increased up to 7 g/L, approximately 62 wt % of which corresponds to mature human insulin. A two-stage fed-batch fermentation process, with recombinant cell growth occurring at a constant growth rate and constant cell concentration in a growth fermenter and mini-proinsulin production in an induction fermenter, is designed, and its efficacy in increasing volumetric productivity of mini-proinsulin is demonstrated.