[Prokaryotic expression, purification and identification of recombinant Xenopus laevis and mouse vascular endothelial growth factors].
OBJECTIVE
To perform prokaryotic expression, purification and identification of Xenopus laevis and mouse vascular endothelial growth factors (VEGF).


METHODS
The prokaryotic expression vectors were constructed by restriction endonuclease digestion and ligation, then the recombinant vector pET-xVEGF and the control vector pET-mVEGF were identified by restriction enzymatic digestion and DNA sequencing. The confirmed vectors were transformed into Escherichia coli. BL21 (DE3) and recombinant protein expression was induced by Isopropy-beta-D-thiogalactoside. The recombinant xVEGF and mVEGF were obtained and purified by Ni-NTA affinity chromatography under denature conditions and enterokinase specific digestion, respectively. Finally, the purified proteins' molecular weights and specificity were detected by SDS-PAGE and western blot analysis.


RESULTS
Two new recombinant expression vectors, pET-xVEGF and pET-mVEGF, were constructed successfully. The xVEGF and mVEGF fusion proteins were expressed in E. coli. BL21 (DE3) stably, and the molecular weights of the purified xVEGF and mVEGF were identical to the expected values. The purity of final products reached a level higher than 95%. In addition, these two purified proteins could react with a specific antibody against mouse VEGF as expected.


CONCLUSIONS
Recombinant Xenopus laevis VEGF and its control mouse VEGF protein may provide tools for further study of anti-tumor active immunity with this xenogeneic protein vaccine in mouse tumor models.
Niu
Ting
T
The State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China.
Liu
Ting
T
Jia
Yong-qian
YQ
Yang
Li
L
Tian
Ling
L
Liu
Ji-Yan
JY
Hu
Bing
B
Wu
Yang
Y
Wei
Yu-quan
YQ
D000818:Animals; D019496:Cancer Vaccines; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D051379:Mice; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014982:Xenopus laevis
D019496:Cancer Vaccines; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A