Expression of extracellular domain of human vascular endothelial growth factor receptor-2 in Pichia pastoris.
OBJECTIVE
To investigate the feasibility of high expressing extracellular domain of human vascular endothelial growth factor receptor-2 (heVEGFR-2) with eukaryotic protein structure in Pichia pastoris.


METHODS
We used PCR to amplify the DNA fragment encoding heVEGFR-2 from pORF-heVEGFR-2. The recombinant Pichia pastoris secretory expression vector(pPICZalphaA-heVEGFR-2)was constructed and transferred into Pichia pastoris X-33 by electroporation. The high expression transformnants were identified through its drug-resistant phenotype and methanol induction.


RESULTS
As indicated by SDS-PAGE, the recombinant heVEGFR-2 protein with a molecular weight (MW) approximately 108 kDa, which reached 80 mg/L in the mass concentration, comprised 45% of the total expressed secreted proteins from Pichia pastoris X-33. The section of heVEGFR-2 had a MW approximately 106 kDa. The results of western blot analysis demonstrated that this protein could be specifically recognized by the rat monoclonal antibody against mouse VEGFR-2 (rat McAb against mVEGFR-2).


CONCLUSIONS
The heVEGFR-2 with eukaryotic protein structure can get a high expression in Pichia pastoris.
Division of Cancer Biotherapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, China.
Qiu
Q
Zuo
Ling
L
Tian
Jian-mei
JM
Hou
Yong-sheng
YS
Wang
Yan-jun
YJ
Wen
Jiong
J
Li
Yu-quan
YQ
Wei
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection / Q000379:methods; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2