[Gene expression and activities analysis of a new fusion protein (RGD) 3/tTF].
To develop a new fusion protein (RGD)3/tTF for the therapy of the selective thrombosis of tumor blood vessels. The fused gene (RGD) 3/tTF was reconstructed by PCR, was cloned into vector pET22 b(+), and expressed in E. coli BL21 (DE3). The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was detected by clotting assay and F X activation assay. The specific binding of (RGD) 3/tTF to alphavbeta3 was analyzed by indirect ELISA. The recombinant plasmid pET22 b(+)/(RGD)3/tTF was obtained and expressed in E. coli BL21 (DE3). The purified fusion protein could induce blood coagulation, activiate F X. The ability of (RGD) 3/tTF binding specifically to alphavbeta3 was increased by 32%, compared with RGD/tTF. A new fusion protein (RGD) 3/tTF was successfully expressed in E. coli BL21 (DE3). The expressed proteins retained tTF activity and showed a higher binding to alphavbeta3 than that of RGD/tTF.
Yan
Jiang-Hua
JH
Cancer Research Center of Medical School, Xiamen University, Xiamen 361005, China. jhyan@xmu.edu.cn
Yang
Gui-Wang
GW
Wang
Jie-Ping
JP
Wu
Na
N
Zhuang
Guo-Hong
GH
D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects*; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism
D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C526680:tTF-RGD peptide; C047981:arginyl-glycyl-aspartic acid; D013925:Thromboplastin; D015951:Factor Xa