A GENETICALLY-ENGINEERED SINGLE-CHAIN FV TNF MOLECULE POSSESSES THE ANTITUMOR IMMUNOREACTIVITY OF FV AS WELL AS THE CYTOTOXIC ACTIVITY OF TUMOR-NECROSIS-FACTOR/

Recombinant DNA techniques were used to clone, construct and express t he fused gene FV-TNF in E. coli under control of the strong T7 bacteri ophage promoter in the expression vector pT7-7-FV-TNF. The fusion prot ein FV/TNF in inclusion bodies from the bacteria homogenate was solubi lized in the denaturing solution containing 6 mol/l guanidine and 0.3 mol/l DTT and refolded in refolding buffer containing 8 mmol/l GSSG. T he FV/TNF was purified by ion exchange chromatography. The yield of FV /TNF was estimated at 10 mg/l. The purified FV/TNF displayed a single band of 42 kD under reducing conditions, whereas it showed three forms including its monomer (40/42 kD), its dimer (84 kD) and its trimer (1 26 kD) under non-reducing conditions. Our data showed that this fusion protein retained its bifunctional activities well, namely the anti-TA G72 immunoreactivity of the FV portion and the cytotoxic activity of t he TNF moiety. Therefore, the FV/TNF fusion protein may prove useful i n targeting the biological effect of TNF to tumor cells as well as in stimulating the immune destruction of tumor cells.