Expression of Tumstatin183-230-TRAIL fusion protein and identification of its biological functions
10.3724/SP.J.1008.2008.00474
- Author:
Na REN
1
Author Information
1. Department of Biochemistry and Molecular Biology
- Publication Type:Journal Article
- Keywords:
Biology activity;
Fusion protein;
TRAIL;
Tumstatin183-230
- From:
Academic Journal of Second Military Medical University
2010;29(5):474-478
- CountryChina
- Language:Chinese
-
Abstract:
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.
