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Expression of human vascular endothelial growth factor (VEGF165) in Pichia pastoris and its biological activity
1Institute of Molecular Immunology, First Military Medical University, Guangzhou 510515, China.
Objective:
To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it.
Methods:
By inserting hVEGF165 cDNA coding 165 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 was expressed under the induction of 1% methanol.
Results:
SDS-PAGE showed that after being induced with 1% methanol for 4 d, 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 Heparin-Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC.
Conclusion:
hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.