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  • Title: [Prokaryotic expression, purification and identification of recombinant Xenopus laevis and mouse vascular endothelial growth factors].
    Author: Niu T, Liu T, Jia YQ, Yang L, Tian L, Liu JY, Hu B, Wu Y, Wei YQ.
    Journal: Sichuan Da Xue Xue Bao Yi Xue Ban; 2005 May; 36(3):301-4. PubMed ID: 15931851.
    Abstract:
    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. CONCLUSION: 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.
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