Akta purifier system

SCX chromatography using the AKTA Purifier system (GE Healthcare) was used to fractionate the iTRAQ labeled peptides. After being reconstituted and acidified with 2 mL buffer A (10 mM KH₂PO₄ in 25% of ACN, pH 2.7), the peptides were loaded onto a PolySULFOETHYL 4.6 × 100 mm column (5 μm, 200 Å, PolyLC Inc., Maryland, U.S.A.). Then, the peptides were eluted at 1 ml/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 2.7) for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50–100% buffer B for 5 min. The elution was monitored by absorbance at 214 nm, and the fractions were collected after every 1 min. The collected fractions (~30 fractions) were combined into 10 pools and desalted on C18 Cartridges [Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma]. Each pooled fraction was concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v/v) trifluoroacetic acid and stored at −80°C for LC-MS/MS analysis.

Calf serum (10 ml) was added to 15 ml EtOH (60% v/v) and the mixture was centrifuged for 15 min. The collected supernatant was evaporated and suspended in 8 ml Milli-Q water (fraction [fr.] II). Fr. II with formic acid (0.1%) was applied to 10 ml of ODS resin (Waters Corporation, Milford, MA, USA). After washing with 25% EtOH and then 50% EtOH, the resin was eluted with 75% EtOH containing 0.1% formic acid. The eluted sample was evaporated and dissolved in 0.8 ml buffer A (10 mM phosphate buffer [pH 7.1], 6 M urea; Fr. III). Fr. III was applied to the Superose 12 10/300 GL gel filtration column (GE Healthcare) and eluted with the AKTApurifier system (GE Healthcare). The elution was performed in buffer A at a flow rate of 0.5 ml min⁻¹ and 60 fractions (0.5 ml per fr.) were collected in 2-ml tubes. All fractions, except for Fr. I, were dialysed in 10 mM phosphate buffer (pH 7.1) before assessment of their activities. Protein concentrations were measured using Coomassie Plus^TM protein assay reagent (Thermo Fisher) with bovine serum albumin as the standard. Amino acid sequence analysis was performed by in-gel digestion and mass spectrometric analysis^{45 (link)}. One unit was defined as the activity that decreases the MIC of lysocin E to 1 µg ml⁻¹.

The enzymes were purified with an AKTA purifier system (GE Healthcare, Sweden). The cell pellets of the overexpression product were resuspended in binding buffer (20 mM sodium phosphate, pH 7.6). The solution was supplemented with 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication. The cell debris was removed by centrifugation at 4 °C at 15,000×g for 10 min. The purified enzyme solution was filtered (0.22 μm filter) before loading onto a 1 mL of HiTrap Capto Q anionic chromatography column and then washed with 10 mL of binding buffer. The enzyme was eluted with an elution buffer (20 mM sodium phosphate, 1 M NaCl, pH 6.0). The fraction containing the target enzyme was loaded onto a HisTrap HP affinity column for further purification as previously described [33 (link)]. Enzyme purity was monitored by SDS-PAGE analysis. Enzyme concentrations were measured using the Bradford method (Coomassie Brilliant Blue G-250 method) with bovine serum albumin as the standard [34 (link)].

We performed gel filtration experiments for bOBP and its mutant forms in a buffered solution without and with addition of GdnHCl using a Superdex-75 PC 3.2/30 column (GE Healthcare, Sweden) and an AKTApurifier system (GE Healthcare, Uppsala, Sweden). The column was equilibrated with the buffered solution or GdnHCl at the desired concentration, and 10 µl of the protein solution prepared under the same conditions was loaded on the pre-equilibrated column. The change in hydrodynamic dimensions for the studied proteins was evaluated as a change in the bOBP or the mutant protein elution volume. Multiple proteins with known molecular masses (aprotinin (6.5 kDa), ribonuclease (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa) and conalbumin (75 kDa), which are chromatography standards from GE Healthcare) were used to generate the calibration curve.

Peptides were fractionated by SCX chromatography using an AKTA Purifier system (GE Healthcare). The dried peptide mixture was reconstituted and acidified with 2 ml of buffer A (10 mM KH₂PO₄ in 25% of acetonitrile (ACN), pH 2.7) and loaded onto a Poly SULFOETHYL 4.6 × 100 mm column (5 µm, 200 Å; PolyLC Inc, Maryland, USA). The peptides were eluted at a flow rate of 1 ml/min using a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 2.7) for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50%–100% buffer B for 5 min. The eluents were monitored by absorbance at 214 nm and fractions were collected every 1 min. The collected fractions (approximately 30) were combined into 25 pools and desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml; Sigma Aldrich, St. Louis, MO, USA). Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80 °C until LC-MS/MS analysis could be conducted.

SEC was performed using an AKTA Purifier system (GE Healthcare, Buckinghamshire, England). A HiLoad 16/600 Superdex 200 column (GE Healthcare) was equilibrated with cell lysis buffer. The column was calibrated using a gel filtration calibration kit (GE Healthcare). Each standard protein was dissolved in cell lysis buffer and chromatographed on the column separately. The filtered protein samples were then fractionated on the column (1.0 ml/min; 2 ml/fraction). For immunoblot analysis, proteins in fractionated eluent were enriched with 10 µl StrataClean resin and harvested in 30 µl of 2 x loading buffer.

Gel-filtration chromatography was performed with the AKTA purifier system (GE Healthcare, Chicago, IL, USA) controlled by the Unicorn 5.1 software. The running buffer contains 20 mM NaCl, 25 mM HEPES, and 0.2 mM 2-mercaptoethanol. Superdex 75 10/300 GL column (Cytiva) with a void volume (V_o) of 7.2 mL was used. The calibration curve was obtained by plotting the molecular weight of marker proteins against the partition coefficient (K_av). K_av was calculated with the equation: K_av = (V_e-V_o) / (V_t-V_o). V_e: elution volume; V_o: void volume; V_t: total volume.