Human epidermal growth factor (hEGF) was expressed in Escherichia coli BL21 under the control of PL promoter and was secreted by using the alkaline phosphatase signal sequence. In batch cultivations, temperature was shifted up from 30 degrees C to one spanning 38-42 degrees C, and delay of the post-induction cell growth was observed, which resulted from expression of the cloned gene. The overall hEGF expression was enhanced with the increase in the induction temperature, while the ratio of extracellular to total hEGF showed an opposite trend. The highest ratio of extracellular hEGF was achieved when induced at 38 degrees C; about 45% of the total hEGF produced was secreted into the medium. Therefore, an induction temperature of 38 degrees C was adopted. In fed-batch cultivations, the pre-induction nutrition condition had a great influence on the initial hEGF productivity after the temperature up-shift, and supply of glucose at a constant specific rate of 0.196 g g(-1) h(-1) improved hEGF production and secretion. By using a rich medium, a final cell density of 26.6 g DCW l(-1) and extracellular hEGF production of 380 mg l(-1) were obtained. (c) 2006 Elsevier Inc. All rights reserved.