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

Abstract

SummaryThe anti-angiogenic activity of tumstatin45-132 is mediated by binding to a V b 3 on endothelial cells and tumor vascular endothelium showing increased expression of a V b 3 . Tumor necrosis factor a (TNF-a) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-a as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-a 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-a activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with antiintegrin antibodies showed that the tumstatin45-132 moiety specifically interacts with a V b 3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo.

Endogenous Transmembrane TNF-Alpha Protects Against Premature Senescence in Endothelial Colony Forming Cells

“…Interestingly, in a previous study we observed a pronounced upregulation of tmTNF in angiogenic tumor blood vessels 32 , which is in line with studies demonstrating involvement of endothelial progenitors in tumor angiogenesis, a process also referred to as vasculogenesis 34-36 . In contrast to the proposed maintenance function of tmTNF in ECFC in vascular repair and in angiogenesis, soluble TNF-α is predominately associated with inflammation, vascular dysfunction, and impaired repair 26 , and according to our group and others acts overwhelmingly through TNFR1 in endothelial cells 13 . Our data reported here show that removal of either tmTNF or TNFR2 causes ECFCs to lose their proliferative potential and develop premature senescence, which provides a mechanism for the observed role of TNFR2 in angiogenesis and vascular repair.…”

“…18, 19, 26 Because of the known role of ECFCs in angiogenesis we examined the role of tmTNF-α in ECFC proliferation. We separated MNCs from cord blood into tmTNF positive and negative fractions by magnetic bead separation.…”

Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution

“…Furthermore, the oligomerization of the VAS domain may result in higher binding avidity to endothelial cells and cause enhanced inhibitory effects on the FBHE endothelial cells compared with vasostation [33]. Thus, our study demonstrated a successful refolding strategy that efficiently achieved high recovery of the protein VAS-TRAIL, which showed promising therapeutic potential.…”

Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli

“…Materials pMD-18T/tumstatin, which contains the full cDNA sequence of tumstatin, was previously constructed and maintained in our laboratory [8]. Expression vector pMAL-c2 and Amylose Resin Heads were purchased from New England Biolabs.…”