Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor α over-expressed in Escherichia coli

A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor α (TNFα) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFα, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of αvβ3 and αvβ5 integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1 l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.