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Expression of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain 1-3 Loop cDNA in Pichia pastoris, Purification of the Expressed Product and Detection of Its Biological Activity

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Biomedical and Environmental Sciences 2001年第4期14卷 292-301页

作者： MALI ZHANGZHI－QING 等 InstituteofMolecularImmunology ThefirstMilitaryMedicalUniversityGuangzhou510515China statekeylaboratoryofmolecularvirologyandgeneticengineering Beijing100052China

Objective To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity. Methods By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His + Mut s phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. Results SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF 165 and inhibit the proliferation of HUVEC stimulated by hVEGF 165. Conclusion Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

关键词： Base Sequence Blotting Western Cells Cultured Cloning Molecular DNA Primers DNA Complementary Electrophoresis Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay Humans Phenotype Pichia Plasmids Polymerase Chain Reaction Proto-Oncogene Proteins Receptor Protein-Tyrosine Kinases Recombinant Proteins Research Support Non-U.S. Gov't Vascular Endothelial Growth Factor Receptor-1

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