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.