人Cu,Zn-SOD在酵母系统中的表达

Intracellular Expression of Human Cu,Zn-SOD cDNA in Pichia pastoris

甲醇酵母 (Pichia pastoris)是一种理想的真核蛋白高水平表达系统。将人 Cu,Zn- SOD基因克隆到酵母整合型质粒载体 p PIC3.5K,经转化 his4缺陷型酵母 GS1 1 5,用 MD培养基及 PCR方法筛选阳性转化子 ,并用含 G41 8的 YPD培养基筛选多拷贝转化子 ,经甲醇诱导表达。 SDS-PAGE和蛋白质印迹杂交结果证实了表达产物为重组的人 Cu,Zn- SOD,经计算机扫描分析 ,表达量占酵母可溶性蛋白的 1 4% ,所表达的人 Cu,Zn- SOD比活可达每毫克蛋白质 77U。

The methylotrophic yeast, Pichia pastoris heterologous gene expression system was utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Human copper, zinc superoxide dismutase (hCu,Zn SOD) gene was cloned into the yeast integrative vector pPIC3.5K , which was then transformed into his4 mutant yeast GS115.PCR fast detection methods was performed to screen the positive transformants and specific medium was used to screen the multiple copy transformants. Via inducing AOX1 promoter by methanol hCu,Zn SOD could be expressed intracellularly. SDS PAGE and western blot were applied to confirm the product.