Figure 3.

Enhanced secretion of Fc-fusion proteins and monoclonal antibodies. (a) Fc-fusion protein with the extracellular domain of Tumor necrosis factor receptor superfamily member 1B (TNFRSF1BTF-Fc) was expressed in HEK293 cells with (+) or without (−) a Flag-tagged protein A (TD^A) or a Flag-tagged J-domain fragment fusion protein carrying protein A as a targeting domain (J^erdj3-TD^A). Two days later, the cell culture medium was harvested, and a western blot assay was performed with anti-human IgG antibody to detect TNFRSF1BTF-Fc. J^erdj3-TD^A in cell lysate was detected by an anti-Flag antibody. GFP was expressed in all samples as a transfection reporter. (b) The same J-domain fragment fusion protein (J^erdj3-TD^A) was expressed with Fc-fusion protein of the V5-tagged Tumor necrosis factor receptor superfamily member 1 A (TNFRSF1A-V5-Fc) in HEK293 cells. TNFRSF1A-V5-Fc and J-domain fusion protein were detected in the cell culture medium and cell lysate, harvested two days after transfection, by western blot analysis using an anti-V5 antibody or anti-Flag antibody, respectively. A reporter plasmid (GFP) was co-transfected to monitor transfection efficiency (lower panel). (c) The dual-expression plasmid expressing both heavy chain and light chain for anti-IL8 antibody was transfected in HEK293 cells with (+) or without (−) a J-domain fragment fusion protein carrying protein A as a targeting domain (J^erdj3-TD^A). Two days later, the cell culture medium was harvested, and antibody production was detected by western blot analysis using an anti-human IgG antibody. The J-domain fragment fusion protein and reporter protein (GFP) was detected in the cell lysate by an anti-Flag antibody and anti-GFP antibody, respectively. (d) The culture media from cells expressing Fc fusion proteins or antibodies were harvested, and their antigen-binding activities were measured by ELISA. Fc fusion proteins and antibody were expressed with (forth bar) or without (second bar) targeting the J-domain fragment fusion proteins (J^erdj3-TD^A). As a control, target proteins were expressed with targeting domain only (TD^A; third bar). The culture medium from control transfectant (empty vector transfection control) was shown in the left bar. Assay plates were coated with the corresponding antigens or binding ligands, and the secreted Fc fusion proteins or antibodies were applied to the respective plates. Bound antibodies or Fc fusion proteins were detected with HRP-conjugated anti-hIgG antibody.