2.2 Gene cloning and protein generation.
A synthetic gene in E. coli codon usage, encoding for the GTN reductase (CAA74280.1), OYE2.6 (XP_001384055), IPR (Q6WAU1) and GDH (WP_003246720), were ordered from Beijing Genomics Institute (BGI, Beijing, China). The synthetic genes were flanked with Nco I andXho I and cloned into the Nco I/Xho I restriction sites of the pET28a vector (NerA and IPR), pET41a vector (OYE2.6), and pET32a vector (GDH) respectively, to generate the expression plasmid pET28a-NerA, pET28a-IPR, pET32a-GDH and pET41a-OYE2.6. The sequence of the resulting expression plasmid was verified by DNA sequencing (Sangon Biotech, Shanghai, China).
Chemically competent E. coli BL 21 (DE3) cells were transformed with pET28a-NerA, pET28a-IPR, pET32a-GDH and pET41a-OYE2.6 and protein expression was performed by standard protocols. Precultures were grown in lysogeny broth (LB) supplemented with kanamycin (50 μg/mL) for NerA, IPR, OYE2.6 and ampicillin (100 μg/mL) for GDH at 37 °C for 12 h and were used to inoculate main cultures (800 mL LB medium containing 50 μg/mL kanamycin or 100 μg/mL ampicillin) to an initial OD600 of 0.05. Cells were grown at 37 °C to an OD600 of 0.6, and protein expression was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM. The cultures were incubated at 35 °C for an additional 10 h, and cells were harvested by centrifugation. Cell pellets were washed with a NaCl solution (0.9%, w/v) and frozen at -20 °C for later purification.