2.3 Expression and purification
Recombinant plasmids, including pET-32a-Ps XR, pET-32a-Rl GDH, pET-32a-Pd PDH, and mutant Pd PDH, were transformed into E. coli BL21 (DE3) cells. Recombinant strains were precultured in 20-mL LB media with 10 μg mL−1 ampicillin at 37 °C and 220 rpm for 24 h. Subsequently, a 20-mL preculture was inoculated into 1-L LB medium (with 10 μg mL−1 ampicillin) at 37 °C and 200 rpm until the optical density at 600 nm reached 0.6–0.8. Then, 0.1 M IPTG was added, and incubation continued at 16 °C with shaking at 180 rpm for 20 h for enzyme expression. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis and His-tagged protein purification were performed as previously described.[13]