Ni2 sepharose histrap hp column

Metagenome-derived genes were PCR-amplified using specific primers (S1 Table) and fosmid DNA as template. PCR products were restricted and inserted in the plasmid pETM10 between NcoI and KpnI. A gene encoding the α-L-fucosidase of T. maritima (Thma) was codon-optimized and synthesized by GeneArt AG (S1 File). The gene was PCR-amplified using relevant primers (S1 Table), restricted and inserted in pETM10 between NcoI and KpnI. The resulting plasmids where used to transform E. coli BL21(DE3) or C41(DE3) strains.
E. coli BL21(DE3) and C41(DE3) harboring recombinant plasmids were cultured in LB medium shaking at 30°C prior to induction at OD₆₀₀ 0.6 with 0.2 mM or 1 mM IPTG. Expression was continued overnight shaking at 25°C. The cell pellets were harvested by centrifugation and re-suspended in binding buffer (20 mM phosphate-citrate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4). Cells were lysed by sonication and centrifuged at 20000 g for 30 min. The supernatant was subjected to sterile filtration through a 0.22 μm filter and subsequently purified using an ÄKTA purifier with Ni²⁺-sepharose HisTrap HP column (5 ml, GE healthcare) mounted according to manufacturer’s recommendations. Protein concentrations were determined using the BCA protein assay (Thermo scientific, USA, Waltham) with BSA as the standard.

The overnight culture of E. coli hosting the Sphingomonas sp. alginate lyase SALy was prepared, as described in Manns et al. 2016 [28 (link)]. The expression was performed in 5 L fermenters in auto-induction media (6 g Na₂HPO₄, 3 g KH₂PO₄, 20 g tryptone, 5 g yeast extract, 5 g NaCl, 0.06% v/v glycerol, 0.05% w/v glucose, and 0.04% w/v α-lactose, pH 7.2) at 20 °C. The cells were collected by centrifugation, re-suspended in cold extraction-buffer (20 mM Tris-HCl buffer, pH 7.4, 250 mM NaCl, 2 mg/mL lysozyme), sonicated to destroy the cell wall, and then centrifuged to remove debris. The supernatant was collected and passed through a 0.22 µm filter, and then loaded onto a Ni²⁺-Sepharose HisTrap HP column (GE Healthcare, Uppsala, Sweden). Unbound material was washed off the column with 10 column volumes of wash buffer 20 mM Tris-HCl, pH 7.4, 250 mM NaCl, 20 mM imidazole. The enzyme was eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 250 mM NaCl, 100 mM imidazole). The enzyme activity of the alginate lyase was ~18 units/mg enzyme quantified as %substrate consumption/(min·mg enzyme) calculated from formation of double bonds at 235 nm on sodium alginate at 40 °C, pH 7 [28 (link)]. SDS-PAGE confirmed protein purity and the protein concentration was determined by the Bradford method [52 (link)].

The soluble fraction of E. coli extracts expressing VapB (pAE-vapB), VapB and VapC (pAE-vapBC) or the solution containing the pressurized VapC (pAE-vapC) were applied to a 1 ml Ni⁺²-Sepharose Histrap HP column (GE Healthcare) previously equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM imidazole. Proteins were eluted using 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 250 mM imidazole and fractions were analyzed by SDS–PAGE, pooled and dialyzed against 50 mM Tris-HCl, pH 8.0. Total protein concentration was determined by Bradford assay.