水稻谷胱甘肽磷脂氢过氧化物酶的表达、纯化及晶体生长条件初筛  

Expression,Purification and Crystal Growing Conditions of Recombinant Oryza sativa PHGPx

作　　者：王丰[1] 刘进元[1] 

机构地区：[1]清华大学生物科学与技术系分子生物学实验室教育部蛋白质科学重点实验室,北京100084

出　　处：《高等学校化学学报》2007年第9期1701-1706,共6页Chemical Journal of Chinese Universities

基　　金：国家'八六三'计划(批准号:2007AA100604);国家重点基础研究发展计划(批准号:2006CB101706);国家自然科学基金(批准号:30170080;39770078);清华-裕元医学科学研究基金资助.

摘　　要：将水稻PHGPx(OsPHGPx)的编码序列克隆到表达载体pGEX-6P-1上,并转化为大肠杆菌进行表达.通过GST亲和层析、离子交换层析和凝胶过滤层析,制备了可用于晶体学研究的OsPHGPx,其纯度超过95%,具备明显的PHGPx活性.质谱显示OsPHGPx的精确分子量为19642.5553,与理论分子量基本一致.OsPHGPx在多个晶体生长条件下出现微晶.三维结构同源建模显示OsPHGPx的结构为硫氧还蛋白折叠形式.Phospholipid hydroperoxide glutathione peroxidase（PHGPx） is a unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. It plays potential important roles in oxidative stress response. Oryza sativaPHGPx gene was cloned into expression vector pGEX-6P-1 and transformed into E. coli strain BI21 （DE3）. OsPHGPx for crystals was prepared with employing Glutathione Sepharose^TM affinity, cation-exchange and gel filtration chromatography. The purity of the purified OsPHGPx was over 95%. OsPHGPx showed an obvious PHGPx activity towards lipid hydroperoxides. MALDI-TOF analysis shows that the exact molecular weight of OsPHGPx was 19275. 568, which was in accordance with the theoretical molecular weight. The microcrystals of OsPHGPx were obtained under several conditions. In addition, a tertiary structure model of the OsPHGPx generated from http：//swissmodel, expasv, org/displayed the thioredoxin fold.

关 键 词：水稻 谷胱甘肽磷脂氢过氧化物酶 表达 纯化 晶体生长 三维结构建模 

分 类 号：O650[理学—分析化学] Q518.2[理学—化学]