Study On Expression And Biological Activity Of Human Vascular Endothelial Growth Factor And Its Receptor Fit-1 Domains In Pichia.pastoris

The vascular endothelial growth factor(VEGF) was identified as endotheiial cell specific mitogen. It has been shown to be involved in endothelial cell proliferation and blood vessel formation. VEGF mediates its function through its specific receptor on the vascular endothelial cell surface. VEGF has important roles in variety physiological and pathological processes including embryogenesis, tumor and some cardiovascular diseases. This paper will focus on three parts below to discuss VEGF and its receptor Fit-1. 1.Expression, purification and biological activity analysis of hVEGF165 in Pichia. pastoris hVEGF165cDNA was amplified from eukaryotic expression vector phVEGF1650SR by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia.pastoris expression vector pPIC9K. Then recombinant plasmid pPIC9K/hVEGF165 was transformed into Pichia. pastoris strains GSI15 and KM7I by both single cross-over and double cross-over methods. Mut phenotype was confirmed and transformants were screened on various concentrations of G418. Multiple insert transformants were selected and fermented in flasks. After four days of methanol induction, recombinant hVEGF165 could be effectively secreted into media, and its highest quantity was estimated to be 60% of total proteins in supernatant and its yield came up to be 0. 3g/L. ELISA and Western blot assays proved it having good ant igenicity and high specificity. Recombinant hVEGF265 was purified by Heparin-sepharose CL6B affinity chromatography, after that its purity came up to 90%. Both HUVEC MTT assay and 3H-thymidine incorporation assay proved that recombinant hVEGF165possessed the natural biological activity of VEGF andwas able to stimulate HUVEC proliferation in a dose-dependent manner. 2. Study on ligand binding domains of human vascular endothelial growth factor receptor Fit-1 with yeast two-hybrid system Four eDNA clones coding truncated Fit-1 mutants consisting of loop2, 1-2, 2-3 and 1-3 were amplified by RT-PCR fromHUVEC. Fusion expression plasmids pGAD424/Flt-1 (2), pGAD424/Flt-1 (1-2), pGAD424/Flt-1 (2-3), pGAD424/Flt? (1?) and pGBT9/hVEGF165 were constructed. Two sorts of plasmids were cotransformed into the yeast reporter strain SFY526. Transformants'β-galactosidase activities were detected by both filter assay and liquid culture assay. The datas showed that Flt?(1?)/hVEGF165 transformant turned blue in two hours in the presence of X-gal and mean β-galactosidase activity was 65. 21U. Fit-1 (2?) !hVEGF165 transformant turned blue in four hours and ft-galactosidase activity was 50. 81U. Fit-1 (1-2) /hVEGF165 transformant took about ten hours and activity was 17. 51U. While Fit-1 (2)/hVEGF165 transformant never turned blue even after thirty hours. It suggested that hVEGF165 binding ability of Fit-1 (2-3) resemble that of Flt-1(1-3), and Fit-1(1-2) had only weak interaction with hVEGF165, while Flt-1(2) failed to bind hVEGF165 by itself. 3. Expression, purification and biological activity analysis of Fit-1 1-2 and 2-3loop in Pickia.pastoris Fit-1 1-2 and 2-3loop eDNA were respectively inserted into pPIC9K and highly expressed in Pichia.pastoris. The expression levels were estimated to be 60% of total proteins in supernatant and the yields came up to be 0. 35g/L. ELISA and Western blot assays proved that recombinant Flt-1(1-2) and Fit-1 1(2-3) had good antigenicity and high specificity.