麻疯树脱氢抗坏血酸还原酶基因克隆及重组蛋白纯化  

Prokaryotic expression optimization and protein purification of Jatropha curcas dehydroascorbate reductase gene(JcDHAR)

作　　者：唐佳[1] 喻川[1] 韩海洋[1] 严君[1] 魏炜[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育重点实验室、四川省生物资源与生物制药重点实验室,成都610064

出　　处：《四川大学学报（自然科学版）》2014年第5期1085-1091,共7页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(NO.31071448)

摘　　要：利用RACE技术克隆出麻疯树脱氢抗坏血酸基因JcDHAR,构建原核表达载体pET-28a-JcDHAR,并对重组菌表达的条件进行了优化,进而进行Ni亲和层析纯化蛋白.结果表明:表达产物与预期大小相符,His-JcDHAR蛋白质的分子质量为30kD;在37℃的条件下0.1mM的IPTG诱导表达5h时,获得了可溶性重组蛋白,通过体外酶学反应鉴定,纯化样品具有酶学活性,表明JcDHAR原核表达成功.The DHAR gene of Jatropha curcas was cloned by RACE technique, was constructed The pro- karyotic expression vector pET-28a-JcDHAR and induced to express in 0. 1mM IPTG and 37℃ for 5 hours to obtain a soluble protein, the molecular weight of expression product was 23.5 kD. Then purified the His-JcDHAR protein by Ni affinity chromatography. The purified target protein was identified by enzymatic reactions in vitro, suggesting that the sample had the enzyme activity and indicating the success of JcDHAR prokaryotic expression.

关 键 词：脱氢抗坏血酸还原酶 原核表达 蛋白纯化 麻疯树 

分 类 号：Q78[生物学—分子生物学]