Structure of the monooxygenase component of a two-component flavoprotein monooxygenase -- Alfieri et al. 104 (4): 1177 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Alfieri et al. 10.1073/pnas.0608381104.

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Protein Preparation.
A. baumannii C₂-hpah gene was cloned in a pET-11a vector (Novagen) and expressed in E. coli BL21(DE3) cells according to the protocol previously described (8, 9). All purification steps were performed at 4°C, and 1 mM DTT was added to all buffers. The cells were resuspended in a lysis buffer [50 mM sodium phosphate (pH 6.8)/5 mM EDTA/0.05 mM PMSF] and disrupted by sonication. The suspension was centrifuged at 34,500 × g for 1 h; the supernatant was added with polyethyleneimine (2.8 mg/g of cells), and centrifuged for 30 min at 17,000 × g. An ammonium sulfate fractionation (20-40% saturated) was performed, and the protein-containing fraction was then resuspended in 50 mM sodium phosphate (pH 6.8) and dialyzed against the same buffer overnight. The sample was then loaded onto a DEAE-Sepharose chromatography column preequilibrated with 50 mM sodium phosphate buffer (pH 6.8) and eluted by a gradient of NaCl (50-300 mM). Then the enzyme was loaded onto a G-200 size-exclusion column equilibrated with 150 mM NaCl in 50 mM sodium phosphate buffer (pH 6.8) and passed through a G-25 column equilibrated with 30 mM Mops (pH 7.0). The purified enzyme does not contain any bound flavin molecule.

For the production of selenomethionine-substituted C₂, E. coli BL21(DE3) cells were transformed with the same vector and grown at 37°C in minimal M9 medium added with 0.1 mM CaCl₂, 1 mM MgSO₄, 0.4% wt/vol glucose, 1.5 mM thiamine, and 50 mg/ml ampicillin until mid-log phase; then they were supplemented with Lys, Phe, Thr (100 mg/liter), Ile, Leu, Val (50 mg/liter), L-selenomethionine (60 mg/liter) and 1 mM IPTG and grown at 25°C for an additional 20 h. Purification procedures were the same as for the native enzyme, except for the addition of 2 mM EDTA in the first steps and 5 mM DTT through the whole process.

8. Chaiyen P, Suadee C, Wilairat P (2001) Eur J Biochem 268:5550-5561.

9. Thotsaporn K, Sucharitakul J, Wongratana J, Suadee C, Chaiyen P (2004) Biochim Biophys Acta 1680:60-66.