[Expression and characterization of fusion protein tTF/SA as a universal effector for targeting blood coagulation].
To prepare a novel fusion protein (tTF/SA) as a universal effector for targeting therapy of blood coagulation and to analyze its biological activities. The fusion gene tTF/SA was constructed by PCR, then inserted into expression vector pET22 b (+), and expressed in E. coli BL21 (DE3). The fusion protein was purified using Nickel-affinity chromatography column. The activities of tTF moiety of the fusion protein were analyzed by clotting assay and FX activation assay, and the binding activities of Streptavidin(SA) to Biotin(B) were analyzed using ELISA.


RESULTS
The recombinant plasmid tTF/SA/pET22 b (+) with the correct sequence was obtained. The fusion gene tTF/SA was expressed with high yield in E. coli BL21 (DE3). The purified fusion protein retain the abilities of activating FX, inducing blood coagulation, and binding Biotin. The fusion gene tTF/SA was successfully expressed in E. coli BL21 (DE3). The recombinant tTF/SA proteins retain the activities of TF and SA. The multitarget therapy of selectively inducing thrombosis in tumor blood vessels can be achieved by the combination of tTF/SA, as a universal effector, and biotinlated carriers directing to tumor blood vessels.
Wang
Zhen
Z
School of Life Sciences, Xiamen University, Xiamen 361005, China.
Yan
Jiang-Hua
JH
Wang
Jie-Ping
JP
Xie
Lian-Ying
LY
Wu
Na
N
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001710:Biotin / Q000378:metabolism; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D013925:Thromboplastin