人Mn-SOD cDNA的克隆及其在巴斯德毕赤酵母中的表达  

Cloning and Expression of Human Manganese Superoxide Dismutase cDNA in Pichia pastoris

作　　者：凌敏[1] 赖祥进[1] 谢科[1] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,南宁530021

出　　处：《生物工程学报》2005年第3期478-481,共4页Chinese Journal of Biotechnology

基　　金：广西自然科学基金资助 (No .9912 0 3 5 )~~

摘　　要：以人肝细胞株(L0 2 )总RNA为模板,用RT PCR扩增出人锰超氧化物歧化酶(hMn SOD)cDNA ,将其插入含有AOX1基因启动子和α分泌信号肽序列的毕赤酵母表达载体pPIC9k ,构建重组质粒pPIC9k MnSOD ,转化毕赤酵母GS115 ,筛选出整合了多拷贝hMn SOD基因的Mut+ 表型菌株,摇瓶培养,0 5 %甲醇诱导表达。SDS PAGE分析显示,诱导4d的培养上清中hMn SOD的表达量约为上清总蛋白的32 % ,酶比活可达2 4 7 7u mg。hMn SOD在巴斯德毕赤酵母中实现了分泌性表达。Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02),and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the α-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut + carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction,the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7u/mg.

关 键 词：人锰超氧化物歧化酶 巴斯德毕赤酵母 分泌表达 

分 类 号：Q786[生物学—分子生物学]