Hitrap sp sepharose column

The cultured supernatant (200 ml) was then collected by centrifugation at 12,000 rcf for 30 min at 4^°C and stored at -80 ⁰C until required. The supernatant was thawed, dialyzed into 50 mM MES buffer (pH 6) and filtered through a 0.45 μm filter before being loaded on to a Hi-trap SP sepharose column (GE Healthcare Life Sciences) pre-equilibrated with 50 mM MES buffer (pH 6) [18 (link)]. Unbound material was removed by washing with equilibration buffer and protein was eluted using a linear gradient of 0–1 M NaCl over 40 ml, with EgKI-1 eluting between 0.4 and 0.6 M NaCl. Purification was monitored by analysis on SDS-PAGE gels and protease inhibitory activity in fractions containing EgKI-1. Purified EgKI-1 was then dialyzed into 50 mM Tris 120 mM NaCl (pH 7) buffer and quantified using the Bradford protein assay [19 (link)]. A sample of the EgKI-1 protein was visualized on 15% SDS-PAGE to verify its purity and the EgKI-1 gel band was subjected to in-gel trypsin digestion and nano high performance liquid chromatography coupled to mass spectrometry (nano LC-MS) [20 (link)].

The yeast supernatant containing secreted brazzein was clarified by centrifugation at 21,600× g for 20 min at 4 °C and by filtration (0.22 µm, Startorius, Göttingen, Germany). Brazzein supernatant was dialyzed in three successive steps against 10 L of 50 mM ammonium acetate, pH 4.0, at 4 °C for 2 days and then loaded onto a HiTrap SP-Sepharose column (5 mL, GE Healthcare Biosciences, Uppsala, Sweden) previously equilibrated with 50 mM ammonium acetate buffer, pH 4.0. The column was washed with 50 mM ammonium acetate, pH 4.0, and elution was performed using an increasing NaCl gradient (from 0 to 1 M). A desalting dialysis was operated against 1 L of 20 mM phosphate potassium adjusted to pH 4.0 using phosphoric acid at 4 °C for 1 day. Under magnetic stirring, the pH level of purified brazzein was raised to 7.5 with 1 M potassium phosphate, pH 7.5. Then, under the same conditions, a second dialysis was performed against 50 mM potassium phosphate pH 7.5. Fractions containing brazzein were concentrated using a Vivaspin concentrator (3 kDa molecular mass cutoff), pooled, and stored at −20 °C. The brazzein concentration was determined spectrophotometrically using an extinction coefficient at 280 nm of 8940 M⁻¹.cm⁻¹.