[Expression of the human extracellular domain of high density lipoprotein receptor in methylotropic yeast].
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 binding, the extracellular domain of HDL receptor was expressed in the methylotropic yeast Pichia pastoris. The DNA fragment encoding for extracellular domain of HDL receptor was cloned by RT-PCR from Hepatoma Bel-7402 total RNA and the nucleotide sequence of the cloned cDNA was verified by inserting it into pGEM-T vector. Then the cDNA was subcloned into pPIC9K, the integrative secretory expression plasmid of Pichia pastoris was constructed, with the methanol-inducible alcohol oxidase promoter and alpha-signal peptide. The recombinant plasmid was transformed into Pichia pastoris 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 band at approximately 64.5 kDa after 5 days of induction. Then the ligand binding activity was confirmed using the DiI-AcLDL as ligand.
Hu
Jian
J
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100050, China.
Hong
Bin
B
Li
Yuan
Y
D000429:Alcohol Oxidoreductases / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C032629:acetyl-LDL; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000432:Methanol