重组人截短型IL-6在大肠杆菌DH5α中的高效表达与纯化

Expression and purification of recombinant truncated human interleukin-6 in Escherichia coli

为研究重组人截短型白细胞介素6(rhIL-6)工程菌高效表达的影响因素及纯化方法的优化。观察不同培养温度对重组人截短型IL-6工程菌生长密度和IL-6表达的影响;复性蛋白终浓度对复性效率的影响;细菌诱导培养后,经破菌-洗包-溶包初步纯化后,再经柱层析纯化IL-6。其结果为30℃培养后42℃诱导培养5h的IL-6表达效率最高,达32.8%;复性蛋白终浓度在0.5g/L以下时,蛋白复性效率最佳,比活性为4.42×108μmol/min·mg-1;进一步柱层析后,IL-6的纯度达96.5%,得率可达46.5%。最后得出培养和纯化工艺简便易行,可获得高纯度、高比活性的截短型rhIL-6的结论。

Purpose to optimize the conditions for expression,renaturation and purification of human recombinant rhIL6 produced in E.coli. Methods: The effects of culture temperature , expression inducing time on final bacterial yield and expression level of IL6 in E.coli were observed; The methods for isolation of inclusion body and renaturation of denatured crude recombinant IL6 were tested;Gel filtration chromatography with Sephacryl S100 was carried out for purification; Bioactivity of resultant rhIL6 was monitored by IL6 dependant cell line 7TD1 proliferative assay. Results The highest bacterial yield and expression rate were obtained (32.6% of total bacterial proteins) at 30℃ of initiative culture and 42℃, 5 hrs of expression induction. Efficient renaturation of IL6 was obtained at 0.5 mg/mL of the denatured recombinant protein. After one step Gelfiltration with Sephacryl S100,the purity of IL6 reached 96.5%, the final yield was 46.5% and specific activity was 4.42×108 μmol/min·mg-1.Conclusion an optimal protocal of fermentation culture, expression and purification of truncated rhIL6 has been established.which is simple, reproducible and practical.