包涵体重组蛋白不同纯化方法的比较Comparison of various methods for purification of recombinant inclusion body protein
常恒祯;常江;战俊澎;杨馨;郭珣;刘益辛;邹德颖;任洪林;
摘要(Abstract)：
目的比较切胶、包涵体复性及Ni-NTA亲和层析3种方法纯化包涵体重组蛋白的效果。方法筛选全长IL-1β与全长IL-1Ra重组质粒(简称IL-1β-1Ra-2重组质粒)及全长IL-1Ra重组质粒的转化感受态菌株[感受态E. coli BL21(DE3)、E. coli BL21(DE3)PLysS及E. coli BL21-Codon Plus(DE3)-RIPL]、IPTG诱导浓度(0.1、0.2、0.4、0.6、0.8、1 mmol/L)、诱导温度(16和37℃)。最佳表达条件诱导的IL-1β-1Ra-2重组质粒,分别用切胶、包涵体复性及Ni-NTA亲和层析法进行纯化,确定最佳纯化方法。采用最佳表达条件分别诱导表达IL-1β-1Ra-2重组质粒及全长IL-1Ra重组质粒后,通过最佳纯化方法进行纯化,纯化产物进行SDS-PAGE及Western blot分析。结果 IL-1β-1Ra-2重组质粒及全长IL-1Ra重组质粒最佳转化感受态菌株为E. coli BL21-Codon Plus(DE3)-RIPL,最佳IPTG浓度分别为1和0.4 mmol/L,诱导温度为37℃;最佳纯化方法为包涵体复性纯化法。IL-1β-1Ra-2及全长IL-1Ra重组蛋白包涵体复性纯化产物相对分子质量分别约为10 560和19 770,纯度可达90%以上,可溶性蛋白回收量分别约为46及63 mg,且均可与小鼠抗His标签单克隆抗体发生特异性结合。结论相同条件下,包涵体复性纯化法获得的可溶性蛋白回收量最大,适用于相对分子质量约10 000的非高表达包涵体蛋白的纯化。
关键词(KeyWords)： 包涵体;纯化;包涵体复性;Ni-NTA亲和层析;切胶
基金项目(Foundation): 国家重点研发计划项目（2018YFD0500900）;; 吉林省科技发展计划项目（20200402059NC）
作者(Authors): 常恒祯;常江;战俊澎;杨馨;郭珣;刘益辛;邹德颖;任洪林;
DOI: 10.13200/j.cnki.cjb.003385
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