Cloning, Expression, and Purification of a Nitric Oxide Synthase-Like Protein from Bacillus cereus

The nitric oxide synthase-like protein from Bacillus cereus (bcNOS) has been cloned, expressed, and characterized. This small hemeprotein (356 amino acids in length) has a mass of 43 kDa and forms a dimer. The recombinant protein showed similar spectral shifts to the mammalian NOS proteins and could bind the substrates L-arginine and NG-hydroxy-L-arginine as well as the ligand imidazole. Low levels of activity were recorded for the hydrogen peroxide-dependent oxidation of NG-hydroxy-L-arginine and L-arginine by bcNOS, while a reconstituted system with the rat neuronal NOS reductase domain showed no activity. The recombinant bcNOS protein adds to the complement of bacterial NOS-like proteins that are used for the investigation of the mechanism and function of NO in microorganisms.

Sequence alignment of the bcNOS protein with prokaryotic and eukaryotic NOS enzymes showed that the residues involved with heme binding in saNOS, bsNOS and the mammalian NOS proteins are conserved in bcNOS. As well, the highly conserved glutamic acid residue found in mammalian NOS proteins to be important for the binding of L-arginine in the substratebinding site is also conserved in bcNOS.
Expression and purification of bcNOS. Escherichia coli strain BL21(DE3) pLysS transfected with the bcNOS plasmid and grown in Luria-Bertani broth containing 100 µg/mL ampicillin, and induced at an OD (600 nm) of 0.6 with 1 mM IPTG for 4 h. The cells were harvested by centrifugation and stored at -80ºC. The temperature was maintained at 4ºC throughout the purification. The harvested cells were resuspended by adding 2 mL/g of cells buffer A (50 mM Tris-HCl buffer pH 7.0, 1 mM β-mercaptoethanol, 1.0 M NaCl) containing 0.1 mg/mL DNaseI and 0.55 mg/mL lysozyme, protease inhibitors pepstatin 2 µg/mL, bestatin 10 µg/mL, chymostatin 10 µg/mL, and leupeptin 25 µg/mL and stirred until the frozen pellet was dissolved. The cells were lysed using a homogenizer and the lysate was centrifuged at 48,000 g.
The cleared lysate was loaded onto Ni 2+ -agarose resin (Novagen, Madison, WI) equilibrated with tris-imidazole buffer containing 1 M NaCl, and eluted with increasing concentrations of imidazole, always in the presence of 1 M NaCl. Fractions were analyzed by SDSpolyacrylamide electrophoresis and Coomassie blue staining, the fractions containing essentially pure bcNOS protein were pooled, dialyzed against 50 mM Tris-HCl, pH 7.5 and 100 mM NaCl and 10% glycerol, The protein was dialyzed over night at 4°C with the dialysis buffer changed once after 3 hours. Protein concentration was determined by the Lowry dye binding microassay method using bovine serum albumin as the standard. All assays were performed in quadruplicate.

Supplementary Figure 2. SDS-PAGE analysis of purified bcNOS expressed in Escherichia coli. Purified bcNOS was electrophoresed on a 10% SDS-PAGE gel (left lane).
Comparison with low molecular weight standard in the right lane shows that the approximate molecular weight for bcNOS is 43 kDa.

Difference spectroscopy
Difference spectroscopy was used to measure the binding affinities of imidazole, NOHA, and L-arginine as previously described [see reference 7 in the manuscript]. All experiments were performed at 25 ºC. The bcNOS enzyme was diluted to a final concentration of 10 µM with 50 mM Tris-HCl, pH 7.5 and 10 % glycerol before titration with imidazole to a final concentration of 10 mM. The additions of imidazole caused < 2% volume changes to the sample. Spectra were recorded from 350 nm to 500 nm after each addition. The apparent binding constant, K d , for imidazole was determined from the x-intercept of a double-reciprocal plot of the difference in the respective peak to trough absorbances versus the imidazole concentration. Linear regression analysis of titration data gave a R 2 value of 0.975. The binding affinities of NOHA and L-arginine were measured after incubation of the bcNOS protein in the presence of 10 mM imidazole for 10 minutes at 25 ºC. Titration of either NOHA or L-arginine caused < 6.5% or < 2% volume changes to the sample, respectively. A double reciprocal plot of the NOHA binding data gave an apparent binding constant, K obs , for NOHA, which was then used to calculate the spectral binding constant, K s , for NOHA using equation 1.
This analysis was also performed on the spectral data obtained by the binding of Larginine to bcNOS in order to determine the K s for L-arginine.