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.