For expression of CoxB in inclusion bodies and the cytoplasmic expression of ScoI and PcuC under T7 promoter/lac operator control, Escherichia coli BL21 (DE3) carrying the corresponding expression plasmid was grown at 37°C in 2YT medium containing ampicillin (100 μg/ml) until an OD600 (optical density at 600 nm) of 0.6 had been reached. Expression was induced by the addition of isopropyl-β-d-thiogalactoside (final concentration, 0.1 mM), and cells were further grown at 30°C for 4 hours and harvested by centrifugation. Proteins were purified according to the following protocols:

CoxB: Cells were suspended in 100 mM tris-HCl (pH 8.0) and 1 mM EDTA (3 ml/g wet cells), mixed with deoxyribonuclease I (DNase I) (50 μg/ml final concentration), and lysed with a microfluidizer (M-110L, Microfluidics, Westwood, MA). After the addition of 0.5 volume of 60 mM EDTA-NaOH (pH 7.0), 1.5 M NaCl, and 6% (v/v) Triton X-100, the lysate was stirred at 4°C for 1 hour. The CoxB inclusion bodies were harvested by centrifugation (30 min, 47,850g, 4°C) and washed five times at 4°C with 100 mM tris-HCl (pH 8.0) and 20 mM EDTA to remove Triton X-100. The inclusion bodies were solubilized in 100 mM tris-HCl (pH 8.0), 6 M guanidinium chloride, 1 mM EDTA, and 100 mM dithiothreitol (DTT) (20 ml/g of inclusion bodies) at room temperature under stirring for 2 hours. Insoluble material was removed by centrifugation (30 min, 142,159g, 20°C). CoxB was refolded at room temperature from the supernatant by rapid, 100-fold dilution with refolding buffer [0.5 M arginine, 1 mM EDTA, 10 mM DTT, and 20 mM tris-HCl (pH 8.0)] and stirring for 1 hour. Refolded and reduced CoxB was concentrated at 4°C to ca. 1.5 mg/ml and dialyzed against 2 × 10 volumes of 20 mM tris-HCl (pH 8.5), 1 mM EDTA, 5 mM DTT, and 1 × 10 volumes of 20 mM tris-HCl (pH 8.5). Precipitated protein was removed by centrifugation, and the supernatant was applied at 4°C to a Resource Q column (GE Healthcare) equilibrated with 20 mM tris-HCl (pH 8.5). CoxB was eluted with a linear NaCl gradient (0 to 1.0 M). Fractions containing pure CoxB according to SDS–polyacrylamide gel electrophoresis (PAGE) were combined; supplemented with 5 mM DTT, 1 mM EDTA, and 5% glycerol; frozen in liquid nitrogen; and stored at −20°C until further use. The final yield of purified CoxB was 60 mg/liter of bacterial culture.

ScoI: Cells were suspended at 4°C in 50 mM acetic acid–NaOH, 1 mM EDTA, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), DNase I (50 μg/ml), and cOmplete protease inhibitor mix (1 tablet/60 ml, Roche Applied Science) (3 ml/g wet cells) and lysed with a microfluidizer (M-110L, Microfluidics). After centrifugation, the supernatant was filtered (0.2-μm pore size) and loaded onto an SP Sepharose column (GE Healthcare) equilibrated with 50 mM acetic acid–NaOH, 1 mM EDTA, and 2 mM DTT. ScoI was eluted with a linear NaCl gradient (0 to 0.5 M). ScoI-containing fractions were pooled, dialyzed at 4°C against 20 mM tris-HCl (pH 9.0), and loaded onto a Source 30Q column (GE Healthcare) equilibrated with the same buffer. ScoI was eluted with a linear NaCl gradient (0 to 0.5 M). ScoI-containing fractions were pooled, concentrated, and loaded onto a Superdex 75 (HiLoad 26/60) column (GE Healthcare) equilibrated with 20 mM tris-Cl (pH 8.5), 0.3 M NaCl, 5 mM DTT, and 0.5 mM EDTA. Fractions containing pure ScoI according to SDS-PAGE were combined, frozen in liquid nitrogen, and stored at −20°C until further use. The final yield of purified ScoI was 30 mg/liter of bacterial culture.

PcuC variants: PcuC and PcuC ΔC were produced as fusion proteins with N-terminal His10 tag, cleavable with tobacco etch virus (TEV) protease. Cells were suspended at 4°C in 50 mM Hepes-NaOH (pH 7.5), 300 mM NaCl, 1 mM PMSF, cOmplete protease inhibitor mix (1 tablet/60 ml), and DNase I (50 μg/ml) (3 ml/g wet cells) and lysed with a microfluidizer (M-110L, Microfluidics). PcuC variants were purified from the soluble fractions of the lysates by application to prepacked Ni2+-NTA (nitrilotriacetic acid) columns (GE Healthcare) equilibrated with 50 mM Hepes-NaOH (pH 7.5), 300 mM NaCl, and 10 mM imidazole. The proteins were eluted with a linear imidazole gradient (10 to 250 mM). PcuC-containing fractions were pooled, and the His10-tags were removed by TEV protease (40 μg/ml; protease/substrate ratio, 1:25) cleavage during overnight dialysis at 4°C against 50 mM Hepes-NaOH (pH 7.5) and 300 mM NaCl. The cleaved His10-tag and TEV protease [His6-tagged variant (58)] were removed by loading the sample onto a Ni2+-NTA column. Flow-through fractions containing PcuC were pooled and incubated with 50 mM EDTA overnight, concentrated, and loaded onto a Superdex 75 (HiLoad 16/60) column (GE Healthcare) equilibrated with 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl. apo-PcuC–containing fractions were pooled, frozen in liquid nitrogen, and stored at −20°C until further use. The final yield of purified apo-PcuC and apo-PcuC ΔC was 26 and 10 mg/liter of bacterial culture, respectively.

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