[Prokaryotic expression, purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli].
OBJECTIVE
To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein.


METHODS
The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.coli expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E.coli BL21 (DE3) pLysS and induced to express fusion protein VEGFR2 D3.4/GST with IPTG. The expressed protein was purified by washing in urea and detected by SDS-PAGE and Western blot.


RESULTS
SDS-PAGE analysis showed that a novel protein with the expected molecular mass (M(r);) about 46 000 was expressed with the inducement of IPTG. And it existed mostly in the form of inclusion body. Grayscale scanning showed that the expressed VEGFR2 D3.4/GST fusion protein accounted for 38.6% of the total bacterium protein. After the purified product was washed by urea, its purity reached 87.1%. Western blot confirmed the recombinant protein was VEGFR2 D3.4/GST fusion protein.


CONCLUSIONS
High purification VEGFR2 D3.4/GST fusion protein is obtained through the E.coli expression system.
Wei
Yu-ying
YY
Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Song
Chao-jun
CJ
Sun
Yuan-jie
YJ
Gong
Jiu-yu
JY
Jin
Bo-quan
BQ
Yang
An-gang
AG
Yang
Kun
K
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D040301:Vascular Endothelial Growth Factor Receptor-2