Akta prime plus fplc

FreeStyle 293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were grown in FreeStyle 293F medium (Thermo Fisher Scientific, Waltham, MA, USA) to a density of 1 × 10⁶ cells/mL at 37 °C with 8 % CO₂ with regular agitation (150 rpm). Cells were transfected with recombinant gp120 expressors using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). One week later, cells were pelleted and supernatants were filtered using a 0,22-µm-pore-size filter (Thermo Fisher Scientific, Waltham, MA, USA). The gp120 glycoproteins were purified by nickel affinity columns, as directed by the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Monomeric gp120 was subsequently purified by fast protein liquid chromatography (FPLC), as previously reported [32 (link)]. The purification by FPLC was performed using an AKTA Prime Plus FPLC with a HILOAD 16/60 Superdex 200 PG (General Electric, Boston, MA, USA). The gp120 preparations (called later in the paper gp120_core) were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use.

All samples were diluted 5-fold with 1X dilution buffer (Tris-Buffered Saline (TBS); 10 mM Tris-HCl with 150 mM NaCl, pH 7.4), filtered using a 0.45 µm pore-size spin filter to remove particulate materials, and centrifuged at 9000× g for 1 min. To deplete Albumin, IgG, α1-Antitrypsin, IgA, IgM, Transferrin, Haptoglobin, α2-Macroglobulin, Fibrinogen, Complement C3, α1-Acid Glycoprotein (Orosomucoid), HDL (Apolipoproteins A-I and A-II), and LDL (mainly Apolipoprotein B)), an AKTAprime plus FPLC (General Electric, Seoul, South Korea) was used to inject 100–200 µL per sample in a Seppro IgY 14 LC5 column (Sigma–Aldrich, St.Louis, MO, USA) with a constant flow rate of 0.5 mL/min for 20 min, followed by a washing step at a flow rate of 2 mL/min for 3 min. Non bound proteins (depleted fraction) were collected in the flow-through fraction. Due to the large volume of collected fractions, depleted samples were concentrated using Amicon™ Ultra-15 Centrifugal Filter Units (Millipore, MA, USA). Concentrated samples were reconstituted in a chaotropic buffer containing 8 M urea, 2 M thiourea, and 100 mM triethylammonium bicarbonate (TEAB) pH 8.5. Concentrated fractions were stored at −80 °C.

Bovine skimmed milk was prepared from raw milk as described previously by El-Fakharany et al.^{48 ,49 (link)}. The pH of skimmed milk was adjusted to 7.0 by adding an equal volume of 0.2 M phosphate, pH 7.0 and applied to CM-Sephadex C50 column pre-equilibrated with 50 mM phosphate, pH 7.0. To remove unbound proteins, column was washed with same buffer and LPO was eluted with gradient of NaCl (0.0 to 1.0 M) in 50 mM phosphate, pH 7.0 at flow rate of 0.5 ml/min and fraction size of 1.0 ml/fraction using AKTA prime plus FPLC (GE Health care, Sweden). After collection and concentration of fractions containing LPO by centricon with cut off 50 kDa, 50 mg protein was applied to Sephacryl S-100 column (16 × 60, GE Health care, Sweden) pre-equilibrated with 50 mM phosphate, pH 7.0. Purified LPO was eluted with 50 mM phosphate, pH 7.0 containing 0.15 M NaCl at a flow rate of 0.7 ml/min and fraction size of 3 ml. The fractions containing LPO were concentrated and desalted by centricon with cut off 50 kDa. The LPO purity was confirmed by 12% SDS-PAGE according to the method of Laemmli^{50 (link)} and native zymogram of LPO was carried out by using 15 mM guaiacol as a substrate in the presence of 3.3 mM H₂O₂ according to methods of Chance and Maehly⁵¹ and El-Fakharany et al.^{52 (link)}.