Expression And Purification Of Vascular Endothelial Growth Factor Receptor-1 In Escherichia Coli

Background:American scholar Folkman the first time give the well-known doctrine:"tumor growth is dependent on tumor angiogenesis".And putting forward a new way-anti-angiogenesis to inhibit tumor growth.Subsequently,it is confirmed by many scholars that tumor angiogenesis play an important role in tumor growth,development,invasion and metastasis.The majority of tumor growth and shift relies on the tumor vascular production.The tumor growth could be classified into non-vascular phase and vascular phase,when the diameter of the tumor reached to 1～2mm, the tumor secrete a number of factors to stimulate vascularization,the tumor which has be supplied blood will increas and transfer.Therefore,a new method for the treatment of tumors is to block tumor angiogenesis.Tumor vascularization is regulated by a number of factors,one of which is the vascular endothelial growth factor(VEGF).So we can see that the Vascular endothelial growth factor has an important significance for curing tumor.It can promote the proliferation of the vessel endothelial cells,and it can promote the transfer of tumor cells too.It exerts biological effects when VEGF combine with Vascular endothelial groth factor receptor.VEGFR-1 works a double of effect to VEGF:It has a negative biological effect on the VEGF in the physiological situation.But VEGFR-1 would mediated the signal transduction in pathological cases,and it plays an important role in the proliferation of the vascular endothelial cell and the elevation of the vascular permeability.Therefore, VEGF and its receptor has become an ideal target for tumor treatment.It can be see that the study of vascular endothelial growth factor receptor (VEGFR)will be of great significance.Objective:Amplification of VEGFR-1 gene,constructing recombinant pET-28a(+)/VEGFR-1,High-level expression and purification of VEGFR-1 protein in E. coli.Methods:The cloning plasmid whith is built in our laboratory is used for the template to design the suitable primer,the PCR amplification product is connected to the plasmid pET-28a(+),The plasmid is named pET-28a(+)/VEGFR-1. Moving the plasmid into DH5αcells,screening of positive strains and extracting the plasmid.Then the plasmid is transferred into the BL21 cells,we can sceen out positive strains.We induced with IPTG to express protein VEGFR-1.The ultrasound is used to break bacteria.The recombinant protein were purified by Ni-NTA agarose column.Results:The recombinant which is named pET-28a(+)/VEGFR-1 is constructed successfully. The expression of the fusion protein is significantly in SDS-PAGE ,and the molecular weight is about 17kD.Conclusion:VEGFR-1 fusion protein with high purity had been obtained after it was eficiently expressed in E.coli.It lays the foundation for anti-cancer drugs and the reseach on protein structure and function.