Akta purifier system

The periplasmic extraction was performed in 100 mM Tris-HCl at pH 7.4, 10 mM EDTA at pH 8.0 containing a protease inhibitor cocktail (Complete EDTA free tablet, Roche, Basel, Switzerland). The mixture was kept overnight at 30 °C in an orbital shaker under constant agitation (250 rpm). After centrifugation at 12,000 rpm for 30 min at 4 °C, the lysate was again clarified by filtration on a 0.2 μm sterile membrane. rhFab_3D1 was purified using a CaptureSelect™ IgG-CH1 affinity column (Thermo Fisher Scientific, Segrate, Italy) according to the manufacturer instructions connected to an AKTA purifier system (Cytiva, Milan, Italy). The Fab was eluted using 0.1 M glycine at pH 2.5. The bound fractions were collected and immediately neutralized with 2 M Tris-HCl at pH 9, buffer exchanged with PBS and concentrated using the Millipore Amicon centricon 30 kDa cut-off (Merck, Darmstadt, German). The oligomeric state of rhFab_3D1 was assessed by gel filtration on a Sephadex 75 column (Cytiva, Milan, Italy) equilibrated with PBS pH 7.0 or 25 mM Tris-HCl, 100 mM NaCl, pH 7.5. The purity was estimated by SDS-PAGE analysis, and the concentration was measured through determination of the absorbance at 280 nm using a NanoDrop 2000C spectrophotometer. The following parameters were used for the calculation: rhFab_3D1 MW 49,714.54 Da; ε_280nm 75,915 M⁻¹ × cm⁻¹).

A detergent screen was performed for the hTRPM4-eGFP fusion protein, where 5 mL pellets of frozen baculovirus infected cells were solubilised in several different detergents including 1% Fos-choline-14, 2% DDM, 1% DM, 1% Lauryl Maltose Neopentyl Glycol (LMNG), 1% digitonin and a mixture of 0.5% DDM plus 0.5% LMNG (wt/vol) dissolved in a base buffer containing (40 mM Tris pH 7.5, 150 mM NaCl, 150 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 6 mM β-mercaptoethanol, 5 mM L-Arginine and 10% glycerol). All cell pellets were solubilised for 2 h at 4 °C with rotation before being subjected to fluorescence size-exclusion chromatography (FSEC) to determine the size homogeneity of the proteins after solubilisation (whether they be aggregated, oligomeric or degraded). FSEC was performed on an AKTA purifier system (Amersham Biosciences) using a Superose 6 10/300 GL column (GE Healthcare) pre-equilibrated with the base buffer and 0.01% (wt/vol) of the detergent the cell pellet was solubilised in. 1 mL of the solubilised material was injected per run onto the Superose 6 column and fractions were collected. Samples from individual fractions (80 μL each) were aliquoted into in a black 96 well microplate (Corning) and scanned for fluorescence using a BMG PHERAstar FS multimode plate reader (BMG Labtechnologies, Durham, NC).

A single colony of Bacillus subtilis BU412 was cultured in YME liquid medium at 32 °C and 160 rpm for 12 h as seed liquid. 3 mL seed liquid was inoculated into 300 mL YME liquid medium and cultured at 32 °C, 160 rpm for 22 h. The supernatant was collected by centrifugation at 4 °C, 16,000×g for 30 min.
The culture supernatant was filtered through 0.22 μm membrane and applied to a Source 15Q 4.6/100 PE column, on an AKTA Purifier system (Amersham Biosciences) pre-equilibrated with 20 mM Tris–HCl (pH 7.5). The column was washed with a linear gradient of 0.5 M NaCl from 0 to 100% concentration in 20 mM Tris–HCl (pH 7.5) at a flow rate of 1 mL min⁻¹. Individual peak fractions were concentrated to 1 mg mL⁻¹ by Amicon ultra centrifugal filters (Millipore) and tested for HR activity on tobacco leaves. Protein samples with HR activity were applied to a Superdex 75 10/300 GL column. The column was eluted with 20 mM Tris–HCl (pH 7.5) at a flow rate of 0.8 mL min⁻¹. Fractions were collected and tested for HR activity, and then determined by SDS-PAGE. All purification steps were performed at room temperature, and the column effluent was monitored by absorbance at 280 nm.

The recombinant strains B. subtilis 168/pMA5‐ido, B. subtilis 168/pMA5‐ido^K152C, and B. subtilis 168/pMA5‐ido^T181C were cultured overnight at 37°C in Lysogeny Broth (LB) medium supplemented with 50 µg/mL kanamycin. The cells were then incubated into 50 mL LB medium and cultured at 37°C shaker at 180 rpm for 20–24 h to obtain IDO, K151C, and T181C protein. The cells were harvested by centrifugation at 8000 rpm at 4°C for 10 min (refrigerated centrifuge, Sigma) and then washed twice with phosphate‐buffered saline (PBS) (50 mM, pH 7.0). The harvested cells were added to lysozyme for 1–2 h on ice and sonicated and centrifuged at 10 000 rpm for 30 min at 4°C to remove cell debris. The supernatant was purified and used for the enzyme activity assay.
Wild type and its variants were subjected to metal affinity chromatography on an AKTA purifier system (Amersham Pharmacia Biotech, UK) using a 1‐mL His Trap FF column (GE Life Sciences, USA) with linear gradient elution experiments using 0 to 700 mM imidazole as the elution buffer. The purified wild type and its mutant enzymes were analyzed by SDS‐PAGE (12% acrylamide). Protein concentrations were determined using a Bradford Protein Assay Kit 32.

The α-amylase fused with polyhistidine tag from recombinant supernatant was purified using immobilized metal affinity chromatography (IMAC) on 5 mL HiTrap IMAC FF, fast flow column with AKTA purifier system (Amersham Biosciences, USA). The column was equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4). Filtered α-amylase supernatant (20 mL) was loaded to the column and the column was washed with the binding buffer. The α-amylase was eluted with the elution buffer (20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4) using a linear gradient of imidazole ranging from 20 to 500 mM. Eluted fractions were collected and assayed for α-amylase and protein. The active fractions were pooled and the homogeneity of the enzyme was determined using SDS PAGE. Aliquots of the purified α-amylase (0.5 mg) in eppendorf tubes were stored at −20°C.

Ixonnexin was loaded onto a Superdex 75 10/300 (GE Healthcare) column equilibrated in 20 mM Tris-HCl, NaCL 0.15 M, pH 8 with a flow of 0.5 mL/min and connected to AKTA purifier system (Amersham Biosciences, Uppsala, Sweden) Pharmacia HPLC system. Protein was detected by peak absorbance at 280 and 220 nm. Column was calibrated with the following recombinant salivary proteins with respective mol wt and retention volumes: D7 (26.7 kDa, 11.64 ml), 9G11 (14.4 kDa, 12.90 ml), FS50 (8.1 kDa, 13.49 ml). The log of mol wt markers was plotted vs the retention volumes resulting in a straight line where retention volumes are interpolated.

The fraction of 20 mg/mL was separated by a Sephadex-G10 column (160 × 70 cm) using an AKTA purifier system (Amersham Pharmacia Biotech, Amersham, UK). The sample was eluted with ultrapure water at 1.5 mL/min and monitored at 214 nm.