Purification of Bifunctional G6PD::6PGL Enzyme

The G6PD::6PGL protein was expressed and purified as previously reported by Morales-Luna et al. [19 (link)]. The E. coli BL21(DE3)Δzwf::kan^r cells containing the pET-3a/g6pd::6pgl vector were induced with 1 mM of isopropyl-β-d-thiogalactoside (IPTG) for 18 h at 25 °C in Luria Bertani (LB) culture medium. The cells were pelleted by centrifugation, suspended in lysis buffer, and disrupted by sonication. Then, by centrifugation (10,000× g for 15 min at 4 °C), the crude extract was obtained and used for protein purification employing a 2′,5′-ADP Sepharose 4B affinity column (GE Healthcare, Chicago, IL, USA) and a Sephacryl 200 (16/60) gel filtration column (GFC) (GE Healthcare, Chicago, IL, USA) [19 (link)]. Purified G6PD::6PGL protein was analyzed in 12% SDS–PAGE gel [20 (link)] and stained with colloidal Coomassie Brilliant Blue (R-250) (Sigma-Aldrich, San Luis, MO, USA). The protein concentration was quantified according to Lowry et al. [21 (link)] using bovine serum albumin (BSA) as the standard.