新型重组IL-6D24-PE40KDEL外毒素融合蛋白复性及纯化

Renaturation and purification of IL-6 D24-PE40KDEL, a novel recombinant IL-6 Pseudomonas exotoxin fusion protein

目的 :对高效表达的重组IL 6D2 4 PE4 0KDEL外毒素融合蛋白进行包涵体的分离、变性与复性以及纯化。方法 :对表达菌体进行超声破碎、洗涤、氧化还原法变性及复性 ,经离子交换层析。结果 :工程菌HB10 1/ pE0 1T的表达产物是以包涵体形式存在 ,菌体的破碎以高渗蔗糖溶液进行预处理后 ,再超声破碎效果最好 ;包涵体的洗涤则先用Triton X 10 0洗 2次 ,再用 8mol/L尿素洗涤一次效果更优 ;最佳变性条件为在 7mol/L盐酸胍中加入0 .0 6 5mmol/L的DTT和 0 .2mol/L的盐酸精氨酸 ;最佳复性条件为在 10℃复性保持 2 4h ,蛋白浓度保持在 30～80 μg/ml,并需加入 0 .2mol/L盐酸精氨酸 ;利用谷胱甘肽的氧化还原法 ,当氧化型谷胱甘肽与还原型谷胱甘肽的浓度比在 2∶1时所获得的活性成分得率最高 ;利用强弱离子交换结合的方法能获得纯度为 95 %的目的蛋白 ,所得融合蛋白可与IL 6及PEA抗体相结合并能特异性地杀伤高表达IL 6R的人多发性骨髓瘤细胞。结论 :本研究获得重组IL 6D2 4 PE4 0KDEL外毒素融合蛋白包涵体的分离、变性与复性条件和一条能获得大量高纯度该融合蛋白的纯化路线。

Objective:To study denaturation,renaturation and purification of IL-6 D24-PE40KDEL. Methods: Ultrasonic wave breaking, renaturation by redox and ionic exchange were used. Results: The protein was expressed in form of inclusion.The bacterial body was broken into pieces by ultrasonic after pretreated with high concentration sucrose. After the inclusion body was washed with 10% triton-X-100 twice and 8 mol/L urea once, the inclusion bodies were denatured by 7 mol/L guanidine hydrochloride containing DTT and arginine hydrochloride, protein was renatured by100 mmol/L tris-HCl,2 mmol/L EDTA(pH 8.0), 0.2 mmol/L arginine hydrochloride,0.004 mol/L glutathione (oxidizing) and 0.002 mol/L glutathione (reduced),10℃,24 hours. The protein was purified by Q Sepharose Fast Flow column,and DEAE Sepharose Fast Flow column. The protein purity was increased to 95% in SDS-PAGE. WB showed that the purified protein could react with IL-6 and PEA antibody. The protein IL-6 D24-PE40KDEL could kill the multiple myeloma cell lines U266 highly expressing IL-6R, but it couldn′t kill the lymphocytic leukemic cell line CEM which did not express IL-6R. Conclusion: A condition of separation, renaturation and purification was gained.