人Mn-SOD cDNA的克隆及其在巴斯德毕赤酵母中的表达

以人肝细胞株(L02)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247、7u／mg。hMn-SOD在巴斯德毕赤酵母中实现了分泌性表达。

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

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 alpha-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 247.7 u/mg.