Expression of human vascular endothelial growth factor (VEGF165) in Pichia pastoris and its biological activity.
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.


CONCLUSIONS
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.
Ma
L
L
Institute of Molecular Immunology, First Military Medical University, Guangzhou 510515, China.
Wang
X N
XN
Zhang
Z Q
ZQ
Chen
A J
AJ
Yao
L H
LH
D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008222:Lymphokines / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors