Expression, purification and biological activity analysis of human vascular endothelial growth factor (VEGF165) in Pichia pastoris

VernacularTitle:VEGF165 cDNA在酵母中的表达、纯化及其生物学活性研究
Author: Li MA ; Xiaoning WANG ; Zhiqing ZHANG ; Aijun CHEN ; Lihong YAO ; Zhongliang JIANG
Publication Type:Journal Article
From: Journal of Cellular and Molecular Immunology 2001;17(1):24-28
CountryChina
Language:Chinese
Abstract: Aim To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expressed recombinant human VEGF165 with good biological activity. Methods Amplifying human VEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptides, the recombinant expression plasmids pPIC9K/hVEGF165 was constructed and transformed into KM71. The multiple insert transformants were screened， fermented in flasks and induced by 10 mL/L methanol. Results After 4 days of methanol induction, the expressed hVEGF165 reached up to 60％ of total proteins in supernatant by SDS-PAGE. Western blot assay proved the expressed hVEGF165 having good antigenicity and high specificity. The recombinant protein was further purified with Heparin-Sepharose CL6B affinity chromatography, and was proved to have good biological activity in stimulating HUVEC proliferation. Conclusion High-level expression of secreted hVEGF165 has been successfully achieved in Pichia pastoris expression system.