重组人α_1-微球蛋白在毕赤酵母中的高效分泌性表达

Secreted expression and purification of recombinant humanα_1 -microglobulin in Pichia pastoris

目的获得可溶、稳定、高纯度的α1-微球蛋白(α1-microglobulin,α1-MG),为临床检测标准品、质控品制备奠定基础。方法制备构建重组人毕赤酵母真核分泌表达质粒,并对重组α1-MG进行诱导表达、纯化和鉴定。应用PCR方法扩增重组人α1-MG pET-15b质粒,得到N端带有6个组氨酸“标签”(6×His-tag)的α1-MG融合基因,将该基因插入到真核酵母分泌表达质粒pPIC9中,筛选得到正确序列的重组分泌表达质粒pPIC9-MG、并转化毕赤酵母菌菌株GS115(Pichia pastoris GS115);用甲醇进行重组人α1-MG的诱导表达,摇瓶发酵,上清α1-MG含量测定;目的蛋白采用硫酸铵沉淀、一步亲和层析纯化并通过免疫学含量测定、PAGE电泳、Western blot以及生物质谱进行鉴定;初步进行稳定性观察。结果成功构建了重组人α1-MG真核酵母表达载体,核酸测序与预测一致,并在毕赤酵母中实现了重组人α1-MG的分泌性表达,表达量可达73 mg／L,约占总蛋白的30％。经纯化及鉴定,得到了可溶性的、具有天然蛋白生物学活性的PAGE纯目的蛋白。结论首次在毕赤酵母中实现重组人α1-MG的高效分泌性表达,表达目的蛋白具有良好的生物学活性、稳定性好、易纯化等优点。

Objective To construct recombinant human α1-microglobulin eukaryotic expression system and make the recombinant protein purify and identify for a stable α1-microglobulin. Methods The recombinant human α1-microglobulin gene with ＂6 × His-tag＂ was amplified by PCR from the recombinant pET-15b-MG plasmid and then inserted into pPIC9 vector. The recombinant pPIC9-MG was transformed into Pichia pastoris strain GS115. Secreted soluble α1-microglobulin was produced by induction of the AOX1 promoter with methanol. The recombinant α1-microglobulin was purified by affinity chromatography and identified by immunoassay, PAGE electrophoresis, Western blot and mass spectrum. Results The recombinant pPIC9-MG expression vector was successfully constructed and secreted soluble recombinant α1- microglobulin was obtained in Pichia pastoris strain GS115. The expression level of the recombinant α1- microglobulin in this strain reached 73mg/L and was about 30% of total secreted protein as judged by SDS- PAGE scanning of protein. The protein was purified by affinity chromatography to PAGE purity. Conclusion The recombinant α1-microglobulin has a high level of secretion in Pichia pastoris yeast and could be recognized by polyclonal antibody of native human α1-microglobulin.