Expression of Tumstatin183-230-TRAIL fusion protein and identification of its biological functions / 第二军医大学学报

Objective: To express Tumstatin183-230-TRAIL fusion protein and to observe its biological functions. Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin183-230 and TNF-related apoptosis-inducing ligand (TRAIL114-281). An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c2; the vector was used to transfect E. coli BL21 (DE3) and expression of MBP-Tu-T fusion protein was induced by IPTG. Amylose Resin columns were employed to purify the fusion protein. The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation, standard tumor cell cytotoxic assay, in vitro tube formation inhibition, and electron microscopic observation (apoptosis). Results: The expression rate of MBP-Tu-T fusion protein in E. coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation (IC50 12.5 μg/ml), induced apoptosis of pancreatic cancer cells, and inhibited tube formation. Conclusion: Constructed MBP-Tu-T fusion protein is bifunctional, which lays a solid foundation for further investigation of antitumor effect of Tumstatin183-230-TRAIL in vivo.

Expression of Tumstatin_(183-230)-TRAIL fusion protein and identification of its biological functions / 第二军医大学学报

Objective:To express Tumstatin_(183-230)-TRAIL fusion protein and to observe its biological functions.Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin_(183-230)and TNF-related apoptosis-inducing ligand (TRAIL_(114-281)).An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c_2;the vector was used to transfect E.coli BL21(DE3)and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein.The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation,standard tumor cell cytotoxic assay,in vitro tube formation inhibition,and electron microscopic observation(apoptosis).Results:The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation(IC_(50)12.5?g/ml),induced apoptosis of pancreatic cancer cells,and inhibited tube formation.Conclusion:Constructed MBP-Tu-T fusion protein is bifunctional,which lays a solid foundation for further investigation of antitumor effect of Tumstatin_(183-230)-TRAIL in vivo.