应用毕赤酵母系统表达人Fascin蛋白  

Expression of Recombinant Human Fascin in Pichia Pastoris

作　　者：李德玲[1,2] 李恩民[2] 谢剑君[2] 吴炳礼[2] 张丕显[2] 许丽艳[1] 

机构地区：[1]汕头大学医学院肿瘤病理研究室,广东汕头515041 [2]汕头大学医学院生物化学与分子生物学教研室,广东汕头515041

出　　处：《癌变．畸变．突变》2008年第6期432-436,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家高新技术研究发展计划(2006AA02A403);国家自然科学基金资助项目(30672376;30772485);广东省自然科学基金重点项目(5104541;7118419)

摘　　要：背景与目的:应用酵母表达系统表达人的重要肿瘤分子标志物fascin蛋白。材料与方法:利用分子克隆技术将fascin基因完整编码区克隆至毕赤酵母(Pichia pastoris)分泌表达载体pPIC9中,获得重组质粒pPIC9-fascin;以限制性内切酶MssⅠ进行线性化并转化毕赤酵母GS115菌株,用PCR方法筛选出阳性整合克隆;对阳性克隆进行0.5甲醇诱导表达重组蛋白,银染及Western blot检测培养上清中fascin重组蛋白的表达情况。结果:毕赤酵母能够分泌表达约69kD的fascin重组蛋白,表达量为40μg/L。结论:毕赤酵母可以分泌表达fascin蛋白,但由于分泌表达量较低,尚达不到工程化要求。BACKGROUND AND AIM： To express the human fascin protein, an important tumor molecular marker, in Pichia pastoris. MATERIALS AND METHODS： The recombinant plasmid pPIC9-fascin was constructed by inserting the full coding DNA sequence of fascin into the secreted expression yeast vector pPIC9. Then pPIC9-fascin was linearized by Mss I and transformed into Pichia pastoris GS115 strain. After identification of transformants by PCR, the positive transformant was cultured and induced by 0.5% methanol. Silver staining and Western blotting were used to analyz the expression level of fascin in the supernatant. RESULTS： Human fascin protein could be expressed in yeast. Silver staining and western blotting analysis from the supernatant showed the molecular weight of fascin protein expressed by the recombinant GS115 strain was about 69 kD, however, the concentration was less than 40 μg/L . CONCLUSION： Mthough human fascin could be expressed by Pichia pastoris expression system, the low level was far off from that required for bioengineering.

关 键 词：FASCIN蛋白 毕赤酵母 分泌表达 pPIC9载体 

分 类 号：Q786[生物学—分子生物学] R735.1[医药卫生—肿瘤]