Soluble-expression, purification and activity analysis of extracellular domain III of flt1.
- Author:
Yinliang XIE
1
;
Yue GU
;
Rui HUANG
;
Xuexia LI
;
Xue DU
;
Jinhong WANG
;
Dongsheng XIONG
;
Chunzheng YANG
;
Yuanfu XU
Author Information
1. The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Endothelial Cells;
cytology;
metabolism;
Escherichia coli;
genetics;
metabolism;
Extracellular Space;
metabolism;
Genetic Vectors;
genetics;
Humans;
Recombinant Fusion Proteins;
genetics;
isolation & purification;
metabolism;
pharmacology;
Solubility;
Umbilical Veins;
cytology;
Vascular Endothelial Growth Factor Receptor-1;
genetics;
isolation & purification;
metabolism
- From:
Chinese Journal of Biotechnology
2009;25(4):580-586
- CountryChina
- Language:Chinese
-
Abstract:
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.
