pCW/MgNPR

Protocol tips

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	E. coli DH5α strains (including pGroEL/ES) transformed with pCW/MgNPR vectors were inoculated into LB medium containing 50 µg/ml of ampicillin and 20 µg/ml of kanamycin. After an overnight incubation at 37oC, the starter culture was seeded into TB expression medium. The expression culture was grown for 4 h at 37oC and then for 24 h at 28oC with shaking at 200 rpm after the addition of 1 mg/ml arabinose, 0.5 µg/ml riboflavin, and 1.0 mM IPTG.

Protocol tips

E. coli DH5α strains (including pGroEL/ES) transformed with pCW/MgNPR vectors were inoculated into LB medium containing 50 µg/ml of ampicillin and 20 µg/ml of kanamycin. After an overnight incubation at 37oC, the starter culture was seeded into TB expression medium. The expression culture was grown for 4 h at 37oC and then for 24 h at 28oC with shaking at 200 rpm after the addition of 1 mg/ml arabinose, 0.5 µg/ml riboflavin, and 1.0 mM IPTG.

Publication protocol

The expression and purification of the M. globosa NPR enzyme were carried out as previously described [17]. In brief, the E. coli DH5α strains (including pGroEL/ES) transformed with pCW/MgNPR vectors were inoculated into LB medium containing 50 µg/ml of ampicillin and 20 µg/ml of kanamycin. After an overnight incubation at 37oC, the starter culture was seeded into TB expression medium. The expression culture was grown for 4 h at 37oC and then for 24 h at 28oC with shaking at 200 rpm after the addition of 1 mg/ml arabinose, 0.5 µg/ml riboflavin, and 1.0 mM IPTG. Bacterial inner membrane fractions containing NPR from M. globosa were isolated and prepared from TB cultures as previously described [17]. The NPR enzyme was purified with a Ni2+ nitrilotriacetate column as previously described [10]. In brief, the membranes were solubilized overnight at 4oC in 100 mM potassium phosphate buffer (pH 7.4) containing 1.0% CHAPS (w/v). The solubilized fraction was then loaded onto a Ni2+-nitrilotriacetate column, and the purified protein was acquired using an elution buffer containing 300 mM imidazole. The eluted fraction containing NPR was subsequently dialyzed at 4oC against 10 mM potassium phosphate buffer (pH 7.4) containing 10% glycerol (w/v) and 0.1 mM EDTA.

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