新型重组IL6D24-PE40KDEL外毒素融合蛋白的表达优化及下游纯化

目的优化提高重组IL6D24-PE40KDEL外毒素融合蛋白在原核细胞中的表达量,并建立有效的下游纯化路线。方法对IL6D24-PE40KDEL的受体菌、菌体生长状态及诱导表达时间等进行优化,经反复筛选确定了高效简捷的纯化路线:即将Q型强离子交换树脂与DEAE弱离子交换树脂相结合的两步纯化法。结果宿主工程菌HB101的优化表达条件为2%接种量,30℃培养3h,42℃再诱导4h,可使目标蛋白的表达量由最初的13.5%提高到23.8%;经12%SDS-PAGE蛋白电泳证实,特异性表达产物为包涵体形式,占包涵体蛋白的40%,分子量约为60kD,与所预测的IL6D24-PE40KDEL融合蛋白的分子量相吻合。两步纯化法可获得纯度>95%的IL6D24-PE40KDEL融合蛋白,纯化蛋白的回收率为8.7%,经Western-blot实验证明,它可同PEA多抗和IL6单抗特异性的结合。MTT试验检测证实,该融合蛋白能特异性的杀伤高表达IL6R的U266多发性骨髓瘤细胞,ID50为180ng/ml;而对IL6R的T淋巴白血病细胞CEM则无杀伤,ID50>1000ng/ml。结论获得了高表达重组IL6D24-PE40KDEL外毒素融合蛋白的菌株并建立了高效简捷的下游纯化路线,融合蛋白能特异性的杀伤表达IL6R的多发性骨髓瘤细胞系U266。

Optimiziation of the expression and purification for a recombinant IL6-Pseudomonas exotoxin fusion protein IL6D24-PE40KDEL

Objective To optimize the expression and purification of the recombinant exotoxin fusion protein IL6D24-PE40KDL.Methods We studied systematically the effects of different host bacterial strains, growth status of bacterial strains and different induction times on the target protein expression and selected E.coli HB101 as the host bacterial strain. After dialysis and filtration, the protein was purified by Q Sephrose Fast Flow column and DEAE Sephrose Fast Flow column.Results The optimum conditions for the expression of IL6D24-PE40KDL were 2% inoculate volume, Ecoli HB101/pIE01T cultured in LB for 3 hours at 30℃,and then induced for 4 hours at 42℃.The protein was expressed in the form of inclusion bodies in which the IL6D24-PE40KDEL content was up to 40%. The expression of IL6D24-PE40KDL was 23.8% of the total bacterial protein. The approximate molecular weight of the protein was 60kD, similar to the theoretical molecular weight. In order to obtain fusion protein with cytotoxic activity, the optimum conditions of capture, washing, denaturation of inclusion bodies were studied. The protein purity was increased to 95%. Immunoblot with antibody showed the purified protein could react with IL6 and PEA antibody. IL6D24-PE40KDEL could kill the multiple myeloma cell line U266 expressing high affinity IL6R with ID50 of 180ng/ml, but could not kill the cell line CEM not expressing IL6R with ID50 over 1000ng/ml.Conclusion A host bacterial cell expressing high level and effective purification of IL6D24-PE40KDEL exotoxin fusion protein have been gained .