白细胞介素-6在工程菌分批培养中的高效表达及其纯化

在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2～3.5g/L之间收菌,rIL-6的表达水平为25％～32％;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70％。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95％以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。

High Expression and Purification of Recombinant Interlukin-6 in E. coli in the Batch Culture

We observed the production of organic acids in the induced procedure of a rising temperature and the effects of the produced organic acids on the growth of the recombinant bacteria E. col: DH5a (pBV-hIL-6) and the expression of rlL-6. The expression of rIL-6 was 25% ~32% when the concentration of organic acids were below 70 mmol/L and cells were harvested at the density of 2 - 3. 5g dry weight]/Lmedium]. The expression of rIL-6 was clearly inhibited when the concentration of organic acids were above 70 mmol/L. while the growth of cell was not affected. Most of the produced organic acid was acetic acid. The purity of rIL-6 from inclusion bodies was about 70% , and the inclusion bodies were isolated from cells by lysis and tracted. After the inclusion bodies dissolved in GuHCl buffer solution were diluted and went through Q Sepharose Fast Flow chromatography, the purity of rIL-6 could reach above 95%. The specific activity of rIL-6 was 2X 108U/mg protein], determined by using the IL-6-dependent mouse-mouse hybrid cell line 7TD1 and MTT colorimetric assay.