Expression, Purification and Biological Activity Analysis of Human Vascular Endothelial Growth Factor (VEGF(165)) in Pichia pastoris.

The human VEGF(165) cDNA was amplified by PCR, and was inserted, after confirming by DNA sequence analysis, into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of alpha factor gene to form a recombinant expression plasmids pPIC9K/hVEGF(165). This recombinant plasmid was transformed into KM71. Transformants were screened, cultured inflasks and induced by the addition of 1% methanol. After 4 days of methanol induction, the expressed hVEGF(165) came up to 60% of total proteins in medium supernatant as shown by SDS-PAGE. Western blot assay proved that the expressed hVEGF(165) had good antigenicity and high specificity. The recombinant protein was further purified by using Heparin-Sepharose CL6B affinity chromatography, and was proved that it had good biological activity to stimulate HUVEC proliferation and to promote collateral blood vessel formation in an acquired limb artery occlusion animal model.