Purification of Recombinant PPX_Δ12 Enzyme

The expression and purification procedure of recombinant PPX_Δ12 was performed as described previously (21 (link)). In brief, DH5α competent E. coli cells were cultured at 37°C on an orbital shaker after transformation with plasmid DNA (pTrcHisB backbone) coding for PPX_Δ12. Induction of protein expression through the pTrc promoter was achieved by addition of 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich). After lysis of cells by sonication, protein purification was performed using an ÄKTA™ start FPLC system (GE Healthcare) connected to a 1 ml HisTrap FF crude column (GE Healthcare). Histidine-tagged proteins were eluted with a buffer containing 20 mM NaH₂PO₄, 500 mM NaCl and 500 mM imidazole. The buffer of the elution fractions was changed to PBS, pH 7.4.