Genetic engineering of a fusion protein possessing anti-tumor Fv and tumor necrosis factor alpha
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In order to target tumor necrosis factor alpha (TNF-α) specifically to tumor cells, we designed, constructed and expressed a recombinant fusion protein single-chain Fv/TNF-α (scFv/TNF-α) containing the scFv of B72.3 monoclonal antibody and the human TNF-α in 'Escherichia coli' using genetic engineering methods. Our efforts have demonstrated that both the TAG72 binding affinity of B72.3 antibody and the biological activities of TNF-α can be transferred to and maintained in the recombinant fusion protein. The recombinant fusion protein was obtained by two means, the 'in vitro' denaturation and refolding of scFv/TNF-α from its inclusion bodies and the secretion expression of scFv/TNF-α. We developed an excretion method for enhanced production of scFv/TNF-α in its secretion form by treatment of 'E. coli' cells with Triton X-100 and glycine to increase the membrane permeability, alleviate the toxicity and prolong the expression. Accordingly, the level of scFv/TNF-α excreted into media was increased by two ordersof magnitude. We also found that some surface exposed hydrophobic residues in the framework region (FR) of scFv had influences on the expression of scFv/TNF-α, in particular, the substitution of a light chain FR residue Phe83 with Ala increased the expression level of scFv/TNF-α by 4 fold. To purify the scFv/TNF-α with TAG72 affinity chromatography, we developed an enzyme-linked immunosorbent assay (ELISA)-elution method for systematically screening elution buffers and identified the optimal elution reagents, one mole ammonia solution, for the elution of scFv/TNF-α from the affinity column. Generally, 1.5 mg of scFv/TNF-α fusion protein could be obtained from one-liter bacterial culture using our expression and purification methods. The antigen binding, antigen-inhibition and antibody-competition ELISAs demonstrated that the scFv/TNF-α retained its bifunctional activities well, namely the anti-TAG72 immunoreactivity of scFv protein and the cytotoxic activity of the TNF-α moiety. Finally, the recombinant fusion protein was able to specifically target TNF-α to tumor cells and tissues expressing the TAG72 antigen in flow cytometry analysis and immunohistochemical studies. Therefore, the scFv/TNF-α fusion protein may prove useful in targeting the biological effect of TNF-α to tumor cell as well as in stimulating the immune destruction of tumor cells.