Purification of Multiple Pyruvate Dehydrogenase Enzymes

AmPDH1, AmPDH2 and AmPDH3 were essentially produced in cultivations in a 7-L laboratory fermenter as described for AmPDH1 (Graf et al. 2015 (link)), using basal salts fermentation medium. A purification scheme as described previously (Graf et al. 2015 (link)) was used. Briefly, this scheme is based on hydrophobic interaction chromatography (HIC) using a Phenyl Sepharose Fast Flow column (GE Healthcare, Little Chalfont, UK), equilibrated with 50 mM potassium phosphate buffer (pH 6.5, 40% saturation (NH₄)₂SO₄), and immobilized metal affinity chromatography (IMAC) with a Ni²⁺-charged Chelating Sepharose Fast Flow column (GE Healthcare), equilibrated with 100 mM potassium phosphate buffer pH 7.0, 1 M NaCl and 5 mM imidazole. An additional ion exchange chromatography (IEX) step using a DEAE-Sepharose Fast Flow column (GE Healthcare) equilibrated with 100 mM potassium phosphate buffer pH 7.0 was added for AmPDH3 as a polishing step.