Objective    To investigate the optimal high cell density fermentation conditions of recombinant E.coli. BL21/pET-MHM to produce recombinant MAGE1/HSP70/MAGE3 fusion protein．    Methods    Based on the experimental results obtained in shake flask cultivation

the cells were transferred to NBS Bioflo 20 L DO feed-back fed-batch culture system. The effects of the composition of the fermentation medium

activation time

induction time

the range of pH and fed-batch carbon sources on the expression level of MAGE1/HSP70/MAGE3 and cell output were analyzed．     Results    By keeping dissolved oxygen at 40%

culturing in semi-defined medium and inducing for 5 h in the presence of 0.2 mmol/L IPTG at 37 ℃ with glycerol as the carbon sources during fed-batch cultivation

we performed a three-time repeated fermentation. The final cell density was D(600) 45 to 50 and more than 70 g of wet bacteria per liter. The expression level of recombinant protein MAGE1/HSP70/MAGE3 was over 38% of the total protein in E.coli.     Conclusion    A Short cycle，high expression and stabilized fermentation process of recombinant MAGE1/HSP70/MAGE3 has been established

which founds the basis for further purification and large-scale production of MAGE1/HSP70/MAGE3.