人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达

研究了hdHGF基因在毕赤酵母中的表达 .以人胎盘mRNA为模板 ,经逆转录、重叠PCR获得hdHGF全长和成熟基因片段 .将该基因片段克隆到pPIC9载体上 ,将重组表达质粒转化巴斯德毕赤酵母 (Pichiapastoris)GS115,筛选mut+ 表型 ,经甲醇诱导可实现rhdHGF的分泌表达 .经摇瓶培养筛选出 4株表达水平较高的酵母工程菌株 ,SDS PAGE分析和Western印迹试验表明 ,产物分子量约为80kD ,5L发酵罐高密度培养已使生物量达 13 5g L(干重 ) ,发酵液上清总蛋白量为 8 0g L ,电泳结果表明rhdHGF表达水平为总蛋白的 12 3 % .

Secretory Expression of the Deleted Variant of Human Hepatocyte Growth Factor(hdHGF) in Pichia pastoris

The expression of hdHGF gene in \%Pichia pastoris\% was investigated.The cDNA encoding the deleted variant of human hepatocyte growth factor(hdHGF) was cloned by RT\|PCR and over lapping\|fragments using mRNA of human placenta as template.The hdHGF cDNA was inserted into plasmid pPIC9 and transformed into \%Pichia pastoris\% GS115 cells.The Mut\ transforments was screened to get high expression strains by the tests of shake\|flask culture.The band of molecular weight of about 80 kD was observed on SDS\|PAGE and idetified by Western blotting.In the 5L high cell density fermentation,the cell biomass reached 135 g/L(DCW),the total supernant protein concentration was 8\^0 g/L,and the rhdHGF band area was about 12\^3% of the total soluble proteins by SDS\|PAGE analysis.