补料分批培养大肠杆菌表达胸腺素α_1融合蛋白

The culture of recombinant E. coli and expression of heterogeneous protein thymosin α_1

在摇瓶培养含有trymosin α1基因的重组pET32a质粒的工程菌BL21表达胸腺素α1融合蛋白的基础上，确定了分批发酵的基本培养条件．分别在B．Braun 5L自控发酵罐中进行了分批发酵和补料分批发酵，针对发酵过程中工程菌生长较旺盛而表达量小的情况，改变了培养方式．比较了不同的培养条件和方式下的工程菌的生长状况和融合蛋白的表达量，得到了高产量和高表达的培养条件：在培养过程中始终保持DO值〉30％，在对数生长期溶氧供给不足的情况下，提高搅拌速度到最大并适当补充纯氧；（2）限制性流加葡萄糖；（3）培养温度35℃，诱导表达温度30℃；（4）对数中期诱导，表达时间为4～5h；（5）在诱导前加入新鲜培养基，稀释发酵液一倍，并以稀释后的发酵液体积为准加入诱导剂．最终使菌体的干重达10g／L，融合蛋白表达量达23．25％．

Fed-batch cultivation of the recombinant E.coli which carries heterogeneous gene coded for thymosin α1 was carried out based on shake flasks using a standard stirred bioreactor with a working volume of 5 L. The cell density and express level of heterogeneous protein were studied on different fermentation conditions. The optimal conditions that achieved are as follows： （1）Keeping on DO level that is higher than 30% by speed increasing and supplying pure oxygen. （2）Fed-batch cultivation in the glucose-limited mode. （3）The temperature suitable for the growth of this recombinant strain and fusion protein expression are 35-37 ℃ and 30℃. （4）The inducing should be started at the middle of exponential growth phase and inducing time lasted 4 - 5 h. （5）Adding to fresh medium before inducing. Finally,a density of 10 g/L was achieved and a high heterogeneous protein expression level of 23.25% is achieved.