人可溶性低密度脂蛋白受体在甲醇酵母中的表达

Expression of the Human Soluble Low Density Lipoprotein Receptor in Methylotropic Yeast

为获得低密度脂蛋白受体配基结合结构域在甲醇酵母中的分泌表达 ,首先用RT PCR方法以人肝癌Bel 740 2总RNA为模板扩增了编码低密度脂蛋白受体配基结合结构域的基因片段。核酸测序分析表明克隆到的DNA片段的序列与报道的人LDLR的cDNA序列相同。然后构建了甲醇酵母表达质粒pPIC9K sLDLr ,并将其线性化后用电穿孔法导入PichiapastorisGS115。分别用SDS PAGE、Westernblot和Ligandbindingblot对GS115 pPIC9K sLDLr上清中的重组sLDLR进行鉴定。SDS PAGE和Westernblot分析表明表达的sLDLR的表观分子量为 36kD。

To obtain the expression of human low density lipoprotein receptor ligand binding domain in methylotropic yeast,firstly the DNA fragment encoding for human low density lipoprotein receptor ligand binding domain was amplified by RT-PCR with human hepatoma Bel-7402 total RNA as template.The nucleotide sequencing analysis indicated that the sequence of the cloned DNA fragment was as same as the reported human LDLR cDNA sequence.Then the expression vector pPIC9K-sLDLr was constructed,linearized and introduced into \%Pichia pastoris\% GS115 by electroporation.The recombinant sLDLR was identified by SDS-PAGE,Western blot and Ligand binding blot in supernatant of GS115/pPIC9K-sLDLr.The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sLDLR was about 36kD.And the ligand binding blot analysis indicated the expressed sLDLR has the biological activity.The sLDLR,which had the biological activity,was successfully secretorily expressed in the Pichia pastoris (GS115).