【摘要】 研究了利用亲和融合谷胱甘肽-S-转移酶(GST)的方法纯化重组人白细胞介素-6(IL-6)的发酵和纯化工艺,使用含有质粒pHZ1818的E.coli JM109在2×YT培养基中进行发酵表达,IL-6表达为与谷胱甘肽-S-转移酶(GST)融合的融合蛋白GST-IL-6。融合蛋白存在可溶的活性蛋白和不可溶的包含体两种形式,此包含体无活性且无法复性,无法用亲和层析回收,因而通过实验优化了摇床发酵的诱导温度和转速以增加可溶融合蛋白的表达。菌体超声破碎液上清液用亲和柱层析,可将融合蛋白提纯至80%,每升发酵液可得到10 mg融合蛋白,用凝血酶裂解处理6 h,亲和标志物GST被特异性切除,裂解得到的IL-6用离子交换柱层析可纯化至95%,MTT法测得纯化的IL-6生物学活性为1.02×10~8 IU/mg。更多还原

【Abstract】 Recombinant E. coli JM109,containing pHZ1818 plasmid which inserted the geng encoding human interleukin-6(IL-6),was expressed as a fusion protein with glutanthion-S-transferase (GST). The fusion protein existed both in the supernatant and pellet of bacterial but the insolube protein had no biological activity and could not be refolded. The rotate speed of shaker and the temperature of induction were studied to optimize the expression of soluble fusion protein. From the supernatant of the cell sonicates a Glutanthion-Sepharose 4B affinity column chromatography was emploved to isolated the fusion protein which could be purified to>80% with a single step. The yield of soluble GST-IL-6 was about 10 mg per liter culture. The GST was site-specifically cleavaged by the thrombin treatment of 6 hours IL-6 was purified by Q high performance ion exchange chromatography from the thrombin digest mixture. From 1 liter E. coli culture 2mg refined IL-6 was obtained. The purified IL-6 had a purity of more than 95% and a biological activity of 1.02×10~8 IU/mg.更多还原