Hu Jian, Li Yuan
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100050, China.
Sheng Wu Gong Cheng Xue Bao. 2002 Jan;18(1):40-4.
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 36 kD. 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).
为了在甲基营养型酵母中获得人低密度脂蛋白受体配体结合域的表达，首先以人肝癌Bel-7402总RNA为模板，通过RT-PCR扩增编码人低密度脂蛋白受体配体结合域的DNA片段。核苷酸序列分析表明，克隆的DNA片段序列与报道的人LDLR cDNA序列相同。然后构建表达载体pPIC9K-sLDLr，线性化后通过电穿孔导入毕赤酵母GS115。通过SDS-PAGE、Western blot和配体结合印迹法对GS115/pPIC9K-sLDLr上清液中的重组sLDLR进行鉴定。SDS-PAGE和Western blot分析表明，表达的sLDLR的表观分子量约为36 kD。配体结合印迹分析表明表达的sLDLR具有生物学活性。具有生物学活性的sLDLR在毕赤酵母（GS115）中成功分泌表达。
