Target cell-restricted apoptosis induction by 528scFv-TRAIL fusion protein specific for human EGFR and expressed in Escherichia coli

We report the preparation and functional charac- terization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFR- positive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a com- mercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.