Prokaryotic expression, purification and activity assay of recombinant vascular endothelial growth factor.
OBJECTIVE
To express human vascular endothelial growth factor (hVEGF(165)) in E. coli JM109 in the form of fusion protein by genetic engineering and test the biological activity and immunological competence of the expressed protein.


METHODS
hVEGF(165) gene was subcloned by PCR and inserted into pQE30 plasmid. hVEGF(165) fusion protein was expressed in E. coli JM109 and purified by Ni(2+)-NTA. The immunological competence of the expressed protein was tested by means of Western blotting and enzyme-linked immunosorbent assay (ELISA), and its biological activity was assayed by chicken chorioallantoic membrane (CAM) and Matrigel angiogenesis assay.


RESULTS
The recombinant hVEGF(165) fusion protein was successfully expressed in E. coli JM109 and its expression accounted for 30% of the total cellular protein. The purified protein presented a single band of 23 kD in SDS-PAGE. Western blotting, ELISA, CAM and matrigel angiogenesis assay showed excellent immunologic competence and biological activity of the recombinant protein.


CONCLUSIONS
Recombinant hVEGF(165) protein with excellent biological activity has been successfully expressed in E.coli JM109, which may facilitate future study in construction of prefabricated tissue-engineered bone graft.
Yang
Pei
P
Department of Orthopedics, Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710004, China.
Wang
Kun-zheng
KZ
Shi
Zhi-bin
ZB
Dang
Xiao-qian
XQ
Yu
Peng-bo
PB
Wang
Chun-sheng
CS
Gong
Fu-liang
FL
D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A