[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.
Ma
L
L
Institute of Molecular Immunology, First Military Medical University, Guangzhou 510515, China.
Zhang
Z
Z
Chen
A
A
D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors