Deae sepharose column

Homogenised membranes were solubilised in a solution of 20 mM Tris pH 8 containing 5 mM CaCl₂ and 2% w/v n-Dodecyl-β-d-Maltopyranoside (β-DDM) in the dark at 4 °C with gentle mixing. Solubilised material was bound to a 50 ml DEAE Sepharose column (GE Healthcare) pre-equilibrated with 3 volumes of buffer A: 20 mM Tris pH 8 containing 5 mM CaCl₂ and 0.03% w/v β-DDM. The column was washed with three column volumes of buffer A containing 130 mM NaCl (WT, Chimeric, Minimal) or containing 100 mM NaCl (Tepidum). Protein was then eluted over a linear gradient from 13 to 30% NaCl (WT, Chimeric, Minimal), or 10–20% NaCl (Tepidum). Peak LH1 containing fractions from each sample were pooled, diluted three fold and rerun on the column as described above. After two-rounds of ion-exchange fractions enriched in LH1 were pooled, concentrated to < 2 ml and further purified by size exclusion on a Superdex 200 16/30 GL column (GE healthcare) in buffer A containing 200 mM NaCl. LH1 containing fractions were pooled, concentrated, aliquoted and stored at − 20 °C until required.

Cell paste containing a co-expression of T4moC and T4moD was re-suspended in 25 mM MOPS, pH 7.5, containing 2% glycerol but no NaCl at a ratio of 1.5 ml per g of wet cell paste31 (link). The cell-free extract was prepared as described above for T4moH. The supernatant was carefully decanted and diluted with two volumes of the above buffer and loaded onto a DEAE Sepharose column (45 mm diameter × 250 mm, GE Health Care) equilibrated in the above buffer and eluted in a 0–450 mM NaCl gradient in the same buffer at a linear flow rate of 5 ml min⁻¹. T4moD fractions were pooled based on activity and T4moC was pooled based on the brown colour of the [2Fe-2S] cofactor. Purity of both proteins was monitored by SDS–PAGE. Pooled fractions were individually concentrated and applied to a Sephacryl S-100 (45 mm diameter × 1,000 mm, GE Health Care) column equilibrated in the above buffer at a linear flow rate of 3.33 ml min⁻¹. Fractions were pooled based on activity and purity, concentrated to ~1 mM for both proteins, and exchanged into 10 mM MOPS, pH 7.5, containing 50 mM NaCl. The purified protein was drop frozen in liquid N₂ and stored at −80 °C.

Plasmid pDNC334A encoding the gene for CYP101 C334A was transformed into Escherichia coli NCM533 cells by electroporation. The C334A mutant of CYP101 is spectroscopically and enzymatically identical to wild-type (WT) and is referred to as such. The only difference is that the C334A mutant does not form dimers in solution, and so is well-suited for NMR experiments44 . Fresh transformants were used to inoculate a 5 mL culture of LB containing kanamycin and chloramphenicol. Cultures were scaled up to 1 L and grown at 37 °C in LB until the OD₆₀₀ reached 0.8. Protein expression was induced with the addition of IPTG to a final concentration of 1 mM. Expression was carried out at 28° for 18 hours. Cells were pelleted by centrifugation at 2220 × g at 4 °C. Pellets were resuspended in 50 mM Tris·HCl, 50 mM KCl, pH 7.4. Cells were lysed by sonication and the extract was cleared by centrifugation at 18000 × g at 4 °C for 35 minutes. The pellet was discarded and the supernatant was filtered through a 0.45 μm filter and applied to a DEAE Sepharose column (GE Healthcare) pre-equilibrated with 50 mM Tris·HCl, 50 mM KCl, pH 7.4. After elution with a linear gradient of 50 mM KCl to 300 mM KCl, fractions with A₄₁₇/A₂₈₀ > 0.4 were combined and used for assays.

T4moF cell paste was re-suspended in 25 mM MOPS, pH 7.5, containing 80 mM NaCl and 2% glycerol at a ratio of 1.5 ml per g of wet cell paste55 (link). The cell-free extract was prepared as described above for T4moH. The supernatant was carefully decanted and diluted with two volumes of the above buffer and loaded onto a DEAE Sepharose column (45 mm diameter × 250 mm, GE Health Care) equilibrated in the above buffer and eluted in an 80–400 mM NaCl gradient in the same buffer at a flow rate of 5 ml min⁻¹. T4moF was pooled based on the colour of the flavin adenine dinucleotide and [2Fe-2S] cofactors and catalytic activity. Purity was monitored by SDS–PAGE. Pooled fractions were concentrated and applied to a Sephacryl S-100 (45 mm diameter × 1,000 mm, GE Health Care) column equilibrated with 25 mM MOPS, pH 7.5, 200 mM NaCl and 5% glycerol at a flow rate of 3.33 ml min⁻¹. Fractions were pooled based on activity and purity, concentrated to ~1 mM and exchanged into 10 mM MOPS, pH 7.5, containing 50 mM NaCl. The purified protein was drop frozen in liquid N₂ and stored at −80 °C.

T4moH cell paste was re-suspended in 25 mM MOPS, pH 6.9, containing 150 mM NaCl and 2% glycerol at a ratio of 1.5 ml per g of wet cell paste31 (link). The cell suspension was sonicated on ice at high intensity for 8 min (15 s on; 30 s off). The supernatant from the sonicated cells was recovered by centrifugation at 39,200 g for 60 min at 4 °C. The supernatant was carefully decanted and diluted with two volumes of the above buffer and loaded onto a DEAE Sepharose column (45 mm diameter × 250 mm, GE Health Care) equilibrated in the above buffer and eluted in a 150–450 mM NaCl gradient in the same buffer at a linear flow rate of 40 cm h⁻¹. Fractions were pooled based on both activity and purity as determined by SDS–polyacrylamide gel electrophoresis (SDS–PAGE. Pooled fractions were concentrated and applied to a Sephacryl S-300 (45 mm diameter × 1,000 mm, GE Health Care) column equilibrated in 25 mM MOPS, pH 7.5, 200 mM NaCl and 5% glycerol at a flow rate of 5 ml min⁻¹. Fractions were pooled based on activity and purity, concentrated to ~800 μM active sites, and exchanged into 10 mM MOPS, pH 6.9, containing 200 mM NaCl. The purified protein was drop frozen in liquid N₂ and stored at −80 °C.

The crude polysaccharide (Component C) was dissolved in deionized water and purified by molecular sieve gel chromatography column (Sepharose CL-4B, 1.6 × 100 cm) using a chromatography system (ÄKTA^TM prime, GE Healthcare, USA). Operating conditions were as follows: the mobile phase was 0.15 moL.L⁻¹ NaCl solution, with a flow rate of 0.2 mL.min⁻¹. The polysaccharide fraction was collected and applied to ultrafiltration. The retentate was further purified by ion-exchange chromatography on a DEAE-Sepharose column (GE Healthcare, USA) equilibrated with 50 mM potassium phosphate buffer (pH 7.5). Elution was performed using a linear gradient of NaCl in the buffer. The collected eluent was applied with active carbon and dialysis (3.5 K MWCO). The purified polysaccharide was finally obtained as a white powder after freeze-drying. Total sugar concentration was determined by the phenol-sulfuric acid method56 (link). The purity of the polysaccharide was measured by gel permeation chromatography (GPC) on a TSKgel G3000PWXL column (7.8 × 300 mm, Tosoh, Japan). The column was eluted with 0.3 M Na₂SO₄ (pH = 4.0) at a flow rate of 0.5 mL.min⁻¹. The eluate was monitored by a refractive index detector (RID).

After ultracentrifugation (150,000 × g, 1.5 h, 4°C), the soluble protein fraction was applied to a DEAE-Sepharose column under anaerobic conditions (50 ml; GE Healthcare) at 5 ml min⁻¹ and washed with buffer A (20 mM Tris/PO₄ [pH 7.0], 1 mM DTT, and 0.02% [wt/vol] Tween 20). The soluble protein fraction was eluted by increasing the amount of buffer B (20 mM Tris/PO₄ [pH 7.0], 1 mM DTT, 0.02% [wt/vol] Tween 20, and 500 mM KCl) from 12% to 16%. Active fractions were concentrated (30-kDa cutoff membrane), diluted in 10 volumes of buffer C (20 mM Tris/morpholineethanesulfonic acid [MES] [pH 6.0], 1 mM DTT), and applied to a Reactive Red 120 column (50 ml; GE Healthcare) at 5 ml min⁻¹. The active fractions were elute with an increasing gradient of buffer D from 0 to 100% (Tris/PO₄ [pH 8.0], 1 mM DTT) in 20% steps. S25DH₁, S25DH₂, S25DH₃, and S25DH₄ eluted at pH values of approximately 6.5, 6.7, 7.5, and 7.8, respectively.