Recombinant PTP1B Expression and Purification

All experiments used the same PTP1B construct as was used previously: residues 1–321, WT* (C32S/C92V double mutation), in the pET24b vector carrying a kanamycin resistance gene (Keedy et al., 2018 (link)). Expression and purification were also performed as previously described (Keedy et al., 2018 (link)). PTP1B was transformed into BL21 Escherichia coli competent cells. The cultures were grown overnight in a 5 mL LB media containing 35 mg/L (final) kanamycin at 37°C shaking continuously at 150 rpm. Next, this overnight culture was used to inoculate 1 L LB media containing 35 mg/L (final) kanamycin. This culture was grown until the optical density at 600 nm (OD₆₀₀) reached between 0.6 and 0.8. PTP1B expression was then immediately induced by adding IPTG to 100 µM (final) and incubating for about 18–20 hr at 18°C shaking continuously at 200–250 rpm. The culture was then pelleted by centrifugation, the supernatant discarded, and the cell pellets (‘cellets’) harvested and stored at –80°C for subsequent purification.