N-terminal 6xhistidine tagged hPAOX, isoform 1 (Q6QHF9-2; Refseq NP_690875.1) was expressed in baculovirus-infected Sf9 insect cells under the AcMNPV polyhedrin promoter (pPolh) transcriptional control. Codon-optimized cDNA was subcloned into pVL1393 (Expression Systems, 91–012) and co-transfected into Sf9 cells with linearized BestBac™ Baculovirus DNA (Expression Systems 91–002). P2 amplified virus was used to infect Sf9 cells for large-scale production in ESF 921 media (Expression Systems, 96-001) for 57 h to final viability of 85% and cell diameter of 21.3 µm.
6His-hPAOX protein was purified by IMAC. Homogenized lysate (10 ml per 1 g cell paste) in 25 mM HEPES, 500 mM NaCl, 20 mM imidazole, 250U/µl Benzonase (Novagen 71205), and 1x protease cOmplete inhibitor cocktail (Roche 05056489001) was bound to HisPur Ni-NTA resin (ThermoFisher) and eluted stepwise in loading buffer supplemented with 40, 80, 160, and 400 mM imidazole.
Pooled fractions from 10 and 40 mM imidazole elutions were yellow, indicating bound FAD co-factor. Pooled Ni-NTA fractions were concentrated with JumbosepTM 10 K MWCO filter (Pall, OD010C65) at 10 °C and loaded onto a Superdex 200 26/600 sizing column (MilliporeSigma) equilibrated with 10 mM HEPES, pH 7.4, 150 mM NaCl. Eluted fractions were pooled and concentrated to 1.47 mg/ml and snap-frozen in liquid nitrogen for long-term storage at −80 °C.
6His-hPAOX protein was purified by IMAC. Homogenized lysate (10 ml per 1 g cell paste) in 25 mM HEPES, 500 mM NaCl, 20 mM imidazole, 250U/µl Benzonase (Novagen 71205), and 1x protease cOmplete inhibitor cocktail (Roche 05056489001) was bound to HisPur Ni-NTA resin (ThermoFisher) and eluted stepwise in loading buffer supplemented with 40, 80, 160, and 400 mM imidazole.
Pooled fractions from 10 and 40 mM imidazole elutions were yellow, indicating bound FAD co-factor. Pooled Ni-NTA fractions were concentrated with JumbosepTM 10 K MWCO filter (Pall, OD010C65) at 10 °C and loaded onto a Superdex 200 26/600 sizing column (MilliporeSigma) equilibrated with 10 mM HEPES, pH 7.4, 150 mM NaCl. Eluted fractions were pooled and concentrated to 1.47 mg/ml and snap-frozen in liquid nitrogen for long-term storage at −80 °C.