[Soluble-expression, purification and activity analysis of extracellular domain III of flt1].
To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Xie
Yinliang
Y
The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Gu
Yue
Y
Huang
Rui
R
Li
Xuexia
X
Du
Xue
X
Wang
Jinhong
J
Xiong
Dongsheng
D
Yang
Chunzheng
C
Xu
Yuanfu
Y
D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D012995:Solubility; D014471:Umbilical Veins / Q000166:cytology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1