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]