在甲醇酵母Pichia pastoris胞内表达有活性的辣根过氧化物酶

为了开辟在甲醇酵母 Pichia pastoris中表达 HRP的新途径 ,将编码成熟 HRP同功酶 C基因克隆到表达载体 p PIK3.5K中 .p PIK3.5KHRP转化 GS1 1 5后 ,用 PCR筛选阳性 P.pastoris重组株 ,并用甲醇进行诱导 . Western印迹杂交分析表明目标蛋白 (约为 38k D)能被天然 HRP的多克隆抗体所识别 ,因此活性辣根过氧化物酶已在 P.pastoris胞内表达 .筛选菌株中显示了明显的过氧化物酶活性 ,同时诱导过程中血红素和 Ca Cl2 的加入对过氧化物酶的活力影响不大

The Intracellular Production of Active Horseradish Peroxidase in Methyltrophic Yeast Pichia pastoris

To explore a new approach to express HRP in methyltrophic yeast, Pichia pastoris ,the gene coding for mature HRP isozyme C was subcloned into the intracellular expression vector pPIC3.5K.After transformation of pPIC3.5KHRP into GS115,the positive recombinant P.pastoris strains were screened and obtained by PCR and induced by methanol.Western blot analysis showed that the target protein(about 38 kD)was recognized by polyclonal antibodies against native HRP.This demonstrated that the active horseradish peroxidase was expressed intracellularly in P.pastoris .The intracellular components of positive recombinant P.pastoris strains showed much higher activity of peroxidase than that of recombinant P.pastoris strain only integrated with pPIC3.5K,and the activity of peroxidase was little affected by the addition of heme and CaCl 2 during induction.