[Expression and characterization of human vascular endothelial growth factor in Pichia pastoris].
OBJECTIVE: To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expression of recombinant human VEGP165 (hVEGF165) with good biological activity. METHODS: Amplifying hVEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and a secreting signal peptides, the recombinant expression plasmid pPIC9K/VEGF165 was constructed and transformed into KM71. The multiple insert transformants were screened, fermented in flasks and induced by 1% methanol. RESULTS: After 4 days of methanol induction, the expressed hVEGF165 came up to 30% of total proteins in supernatant by SDS-PAGE. The expressed hVEGF165 was further proved having good antigenicity and high specificity by ELISA and Western blot assay, and having good biological activity to stimulate HUVEC proliferation. CONCLUSIONS: High-level expression of secreted hVEGF165 had been successfully achieved in Pichia pastoris expression system.[1]References
- [Expression and characterization of human vascular endothelial growth factor in Pichia pastoris]. Ma, L., Zhang, Z., Chen, A. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi (2000) [Pubmed]
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