Expression, Purification
and Biological Activity Analysis of Human Vascular Endothelial Growth Factor
(VEGF165) in Pichia pastoris
MA Li, WANG
Xiao-Ning*
( Institute of Molecular Immunology, The First Military Medical
University,Guangzhou 510515, China )
ZHANG Zhi-Qing*, ZHOU Xiao-Ming, ZENG Ge-Fei, CHEN Ai-Jun
( National Laboratory of Molecular Virology and Genetic Engineering,
Beijing 100052, China )
Abstract The human VEGF165
cDNA was amplified by PCR, and was inserted,after confirming by DNA sequence
analysis, into the Pichia pastoris expression vector pPIC9K containing
AOX1 promoter and lead sequence of α factor gene to form a recombinant
expression plasmids pPIC9K/hVEGF165. This recombinant
plasmid was transformed into KM71. Transformants were screened, cultured
inflasks and induced by the addition of 1% methanol. After 4 days of methanol
induction, the expressed hVEGF165 came up to 60% of total proteins
in medium supernatant as shown by SDS PAGE. Western blot assay proved that the
expressed hVEGF165 had good antigenicity and high specificity. The
recombinant protein was further purified by using Heparin-Sepharose CL6B
affinity chromatography, and was proved that it had good biological activity to
stimulate HUVEC proliferation and to promote collateral blood vessel formation
in an acquired limb artery occlusion animal model.
Key words VEGF； Pichia pastoris； gene expression
*Corresponding author： WANG Xiao-Ning： Tel, 86-20-85148322； e-mail： [email protected]； ZHANG Zhi-Qing： Tel, 86-10-63519655； e-mail： [email protected]