3.2 Recombinant ZnT8/GAD65 expression and purification from cell cultures
The recombinant chimera was expressed in Sf9 cells with a MOI of 1 based on previous experiences (Trabucchi et al., 2019). After 4 days of infection, we efficiently produced ZnT8/GAD65, with a yield of 30 mg of chimera per litre of culture medium, 80% pure. SDS-PAGE analyses of total cell lysates and the different purification steps showed one band of approximately 80 kDa, the expected molecular weight for the full-length engineered chimera (Figure 3). Moreover, the analysis by WB allowed identifying the recombinant chimera using specific antibodies to His6, to GAD65 or to ZnT8.
Even though the purity of the expressed protein obtained is not extremely high, it is suitable for our intended use, the development of highly specific and sensitive immunoassays for the detection of autoantibodies.