Construction, expression and characterization of scFv62-TRAIL. A) Schematic structure of the recombinant fusion construct scFv62-TRAIL, scFv62 (30 kDa) is genetically linked through a flexible Ser-Ser-Gly-Ser-Gly linker to soluble TRAIL (21 kDa). The monomeric scFv62-TRAIL fusion has a molecular weight of 51 kDa and the active trimer a molecular weight of ~150 kDa. B) Stable transfected CHO-K1 cells were used to produce the scFv62-TRAIL fusion protein. Cells were seeded on cell culture flasks, after allowing cell attachment normal medium was change into serum- and protein-free medium and cells were incubated at 37°C or 30°C. The scFv62-TRAIL yield in the medium was analyzed after 5 days by immunoblot using an anti-TRAIL antibody. Under non-reducing conditions scFv62-TRAIL was detected as trimers (150 kDa). C) The scFv62-TRAIL preparation was analyzed using size exclusion chromatography; the received protein peaks were analyzed by immunoblot using an anti-TRAIL antibody. D) Stability of scFv62-TRAIL in mouse serum. The scFv62-TRAIL preparation was incubated in mouse serum at 37°C up to 72 h and analyzed for its ability to induce apoptosis on DU145 cells in the presence of 5 μg/ml CHX (n = 3).