重组人甲硫氨酸硫氧化物还原酶的原核表达、纯化及鉴定  

Expression, purification and identification of recombinant human methionine sulfoxide reductase

作　　者：米苏[1] 王贞[1] 吴洁[1] 喻红[1] 洪嘉玲[1] 李小明[1] 何春燕[1] 

机构地区：[1]武汉大学医学院生化与分子生物学系,湖北武汉430071

出　　处：《医学研究生学报》2004年第4期306-308,共3页Journal of Medical Postgraduates

摘　　要：目的 :表达、纯化并鉴定人甲硫氨酸硫氧化物还原酶 (hMsrA)。　方法 :将含有hMsrA基因的重组质粒转化大肠杆菌 ,经诱导表达 ,表达蛋白主要存在于上清中 ,经破菌、Ni NTAAgarose亲和层析分离纯化目的蛋白。　结果 :SDS PAGE显示纯化的蛋白相对分子质量为 2 6 0 0 0的单一条带 ;Western blot鉴定该纯蛋白有免疫活性 ;酶活性测定表明 ,该纯化蛋白具有还原甲硫氨酸硫氧化物的能力。　结论 :hMsrA能在原核体系中良好表达 ,经纯化后具有催化活性 ,为大量制备并进一步研究抗氧化剂在动脉粥样硬化中的作用提供了科学途径。Objective: To express, purify and identify human methionine sulfoxide reductase (hMsrA).Methods:Recombinant plasmid harboring hMsrA gene was transformed into E. Coli. M15(pREP4). After induction, hMsrA protein was expressed mainly in the supernatant. The protein was purified through high-efficient Ni-NTA agarose column. Results: SDS-PAGE showed that the purified protein was a single band with molecular weight of 26 000. Western blot demonstrated that it reacted specially with anti-human hMsrA antibody. Activity assay further affirmed that the purified protein could catalyze methionine sulfoxide into methionine. Conclusion:hMsrA can be expressed in prokaryotic E. Coli and successfully purified by Ni-NTA agarose column with native activity. It provides a good foundation to purify hMsrA in large scale and further research concerning the functions of anti-oxidation substance in atherosclerosis.

关 键 词：人甲硫氨酸硫氧化物还原酶 Ni-NTA Agamse 纯化 动脉粥样硬化 

分 类 号：R318[医药卫生—生物医学工程]