人高密度脂蛋白受体胞外片段在甲醇酵母中的表达.pdf

　遗 传 学 报 　Acta Genetica Sinica , J anuary 2003 , 30 ( 1) : 20～24 ISSN 0379 - 4172 Expression of the Human Extracellular Domain of High Density Lipoprotein Receptor in Methylotropic Yeast ① HU Jian , HON G Bin , L I Yuan ( Institute of Medicinal B iotechnology , Chinese Academy of Medical Sciences/ Peking Union Medical College , Beij ing 　100050 , China) Abstract : HDL receptor plays a very important role in reverse cholesterol transport ,and it represents a novel therapeutic target for atherosclerosis. For construction of an in vitro high - throughput drug screening model based on competitive receptor - ligand bind ing ,the extracellular domain of HDL receptor was expressed in the methylotropic yeast Pichia p astoris . The DNA fragment encoding for extracellular domain of HDL receptor was cloned by RT - PCR from Hepatoma Bel - 7402 total RNA and the nucleotide se quence of the cloned cDNA was verified by inserting it into p GEM - T vector . Then the cDNA was subcloned into p PIC9K ,the in tegrative secretory expression plasmid of Pichia p astoris was constructed ,with the methanol - inducible alcohol oxidase promoter α ( - ) and - signal peptide . The recombinant plasmid was transformed into Pichia p astoris GS115 His strain by electroporation . PCR was used to confirm the insertion of HDLR gene into the genome of Mut + transformants. During cultivation the recombinant strain was induced by 1 % methanol and supernatant was analyzed by SDS - PAGE and Western blot . The result showed a specific protein