Construction, expression, and characterization of a new targeted bifunctional fusion protein: Tumstatin45-132-TNF

Abstract

The anti-angiogenic activity of tumstatin45-132 is mediated by binding to αVβ3 on endothelial cells and tumor vascular endothelium showing increased expression of αVβ 3. Tumor necrosis factor α (TNF-α) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-α as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-α in the hope of generating a targeting, bi-functional protein in tumor treatment. Tumstatin45-132-TNF was constructed and expressed in E. coli. The recombinant fusion protein was shown to be insoluble and in an inclusion body form. An effective strategy for refolding and purification of tumstatin45-132-TNF resulted in final purified yields of 3 mg purified fusion protein recovered from 1 liter of E. coli culture. The refolded tumstatin45-132-TNF with a purity of 98% assessed by denaturing SDS - PAGE showed a single band on gels. Endothelial cell proliferation assay and standard cytolytic assays against L929 indicated that the fusion protein maintains tumstatin45-132 and TNF-α activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety specifically interacts with αVβ3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo. © 2006 IUBMB.