Figure 2.

Screening method for the detection of effective split SEA designs. A, a schematic diagram of split SEA–coiled-coil fusion constructs used in this study. Three different split SEA variant designs were fused with either P3 or P4 polypeptide through glycine–serine linker (10 amino acids). All constructs were cloned into pFLAG–CMV3 vector containing signal peptide for secretion and FLAG tag for detection of expression. B, schematic representation of newly developed screening method for detection of effective split SEA variant designs. Split SEA fragments are fused with P4 and/or P3 polypeptide and split SEA reassemble into an active form only when split SEA fragments are fused with coiled-coil forming P3 and P4 polypeptides, meanwhile split fragments fused with non–coiled-coil forming P3 polypeptides do not regain its biologic activity. For detection of effective split SEA designs, capable of activating T cells, IL-2 is measured by ELISA. C–E, stimulation assays. Briefly, PBMCs were stimulated with 10 ng/ml of recombinant SEA or with supernatant collected from HEK293T cells containing split SEA–coiled-coil fusion proteins. After 24 h incubation at 37 °C, supernatant was collected and the production of human IL-2, as an indicator of T-cell activation, was measured by commercially available ELISA. PBMCs were stimulated with each split SEA–coiled-coil fusion protein separately (50 μl of each) (C), with combination of split SEA fragments fused with P4 and P3 polypeptide (25 μl of each) (D), and with combination of split SEA fragments fused only with P3 polypeptide (25 μl of each) (E). F, cytotoxicity assay. In brief, the target cell line BCWM was incubated with 25 μl of HEK293T supernatant containing split SEA₂–coiled-coil fusion proteins. SEA-reactive T cell line was used as effector cell line in ratio 1:10. After overnight incubation the percentage of cell lysis of the BCWM cell line was determined by IVIS Lumina. Data are presented as the mean ± S.D. (n = 3), statistical analysis with a two-tailed t test (**, p < 0.01; n.s., not significant).