4Aβ_(15)基因在毕赤氏酵母中的分泌表达及初步纯化  

Secretory Expression of 4Aβ_(15) Gene in Picha pastoris and its Initial Purification

作　　者：谭琳[1] 谭昕[2,3] 周鹏[3] 姚志彬[1] 

机构地区：[1]中山大学中山医学院,中国广东广州510080 [2]海南大学农学院,中国海南海口571101 [3]中国热带农业科学院热带生物技术研究所,中国海南海口571101

出　　处：《生命科学研究》2010年第4期316-321,共6页Life Science Research

基　　金：国家自然科学基金资助项目(30400512)

摘　　要：以pQE4Aβ15为模板,PCR扩增4Aβ15基因,并克隆到pMD18T载体中.以之进一步构建了酵母表达载体pPICZα4Aβ15.通过电转化,重组质粒pPICZα4Aβ15被整合到毕赤氏酵母GS115基因组中.甲醇诱导GS115/pPICZα4Aβ15表达,得到重组蛋白.SDS-PAGE显示重组蛋白的分子质量为18 kD,而4Aβ15的分子质量理论值为8 kD,经Western blot和质谱分析证实重组蛋白为4Aβ15.毕赤氏酵母GS115/pPICZα4Aβ15工程菌发酵上清经60%饱和硫酸铵沉淀初步纯化,获得纯度为80%的重组4Aβ15.Using pQE4Aβ15 as template,4Aβ15 gene was amplified by PCR and cloned into pMD18T vector which was used to construct the yeast expression vector pPICZα4Aβ15.The recombinant plasmid pPICZα4Aβ15 was integrated into genome of P.pastoris GS115 by electroporation.Recombinant protein was obtained after expression in GS115/pPICZα4Aβ15 was induced by methanol.SDS-PAGE analysis revealed that the molecular weight（MW）of recombinant protein is 18 kD,but the theoretical MW of 4Aβ15 is 8 kD.Western blotting and mass spectrum analysis confirmed that the recombinant protein is 4Aβ15.The fermentation supernatant from engineering strain of GS115/pPICZα4Aβ15 was initially purified by 60% saturated ammonium sulfate precipitation,producing 80% purity 4Aβ15.

关 键 词：4Aβ15基因 毕赤氏酵母表达 质谱分析 饱和硫酸铵沉淀 

分 类 号：Q786[生物学—分子生物学]