长效重组人胰岛素原类似物的高密度细胞培养的研究

Studies on high density culture condition for human insulin analogues production in recombinant Escherichia coli

本文以长效重组人胰岛素原类似物(SIIA-0801)的大肠埃希菌工程菌为研究对象,针对采用高细胞密度培养技术表达包涵体重组融合蛋白的关键参数,重点研究了诱导培养基成份(包括碳源、氮源、无机盐、微量元素)、种子细胞密度、IPTG诱导剂的加入量及诱导时间,诱导温度等因素与包涵体表达产量的相关性。结果发现其融合蛋白的最佳表达培养基:1.5%蛋白胨,1%酵母粉,0.5%NaCl,0.8%葡萄糖和0.2%磷酸盐缓冲液(22mmol/LKH2PO4,40mmol/LK2HPO4)。最佳诱导表达条件为:25%接种量,诱导温度37℃,接种培养1h后添加0.5mmol/L IPTG,继续诱导培养5h,发酵培养的总周期为6h。优化后的培养条件比传统的LB培养基及培养方法,其产量提高了6倍多。该结果为长效重组人胰岛素原类似物(SIIA-0801)后续的生产工艺研究提供了重要的参考依据。

An recombinant E. coli expressing long-acting human insulin analogues（ SIIA-0801 ） was used to study the effects of various high-cell-density growth conditions. Our study has revealed that the best growth medium was composed of 1.5% tryptone,l% yeast powder,0.5% NaC1,0.8% glucose,0.2% phosphate buffer solution（22mmol/L KH2PO4, 40mmol/L K2HPO4） and 10μg/ml kanamycin. The optimized culture condition was the addition of 0.5mmol/L IPTG after 25 % inoculation in 1 h with the induction temperature at 37℃, and then continued to ferment for 5 hours. The productivity of fusion protein obtained by the optimized inducing condition and the best growth medium was six times than the traditional induction conditions and the LB medium. All those experiential parameters obtained by the investigation provide the basis for further technology of large scale fermentation of the recombinant E. coll.