Fig. 4

FGE purified from copper(II) sulfate-supplemented cells contains copper and has enhanced specific activity. A gene encoding FGE containing a His₆ affinity tag was cotransfected with CT-tagged antibody into Expi293™ cells. Cells were cultured +/- supplementation with 50 μM copper(II) sulfate. On day 4, antibody was harvested. Then, the cells were washed to remove excess media (and free copper), lysed, and H₆-Hs-FGE was purified from the cell lysate using metal affinity chromatography. Purified FGE was analyzed by reducing and non-reducing SDS-PAGE (a). Then, the purified H₆-Hs-FGE samples were assayed for copper content—by ICP-MS (b)—and for specific activity—via an HPLC-based assay on a peptide substrate (c). Conversion of the antibody was determined by mass spectrometry (d). The experiment was repeated four times with similar results; error bars in C represent standard deviation (n = 4). ICP-MS copper analysis (b) and conversion analysis of the cotransfected antibody were performed once