Hitrap deae column

After cultivation in PDY medium for 4 d, the fermentation broth was harvested by paper filtration and then centrifuged (10,000 g, 20 min). The precipitate formed using 40 to 60% (NH₄)₂SO₄ was collected by centrifugation (20,000 g, 20 min), resuspended in buffer A (25 mM Tris-HCl buffer, pH 7.5), and centrifuged (12,000 g, 5 min). The supernatant was desalted with a HiPrep 26/10 desalting column (GE Healthcare, Buckinghamshire, UK) and applied at 5 mL min⁻¹ to a HiTrap DEAE column (5 mL) pre-equilibrated with buffer A. Adsorbed proteins were sequentially eluted with 0.1 M, 0.2 M and 1 M NaCl in buffer A. Fractions with laccase activity were pooled. (NH₄)₂SO₄ was added until the final concentration was 1 M. The resulting sample was loaded at 1.0 mL min⁻¹ onto a HiTrap Phenyl FF column (5 mL) pre-equilibrated with buffer A containing 1 M (NH₄)₂SO₄. Adsorbed proteins were eluted with a linear 1.0–0 M (NH₄)₂SO₄ gradient in buffer A. Fractions with laccase activity were collected, examined by SDS-PAGE and zymography and stored at 4°C. Deglycosylation of laccase by peptide N-glycosidase F (Takara, Dalian, China) was carried out. Protein identification with MALDI-TOF MS/MS was performed. Protein concentration was quantified by the Bradford method with bovine serum albumin as the standard [24] (link).

To purify the supernatant EPS from the culture of LTC-113 and its derivatives, bacteria was pelleted by centrifugation at 12,000 × g and 4°C for 10 min. Trichloroacetic acid was added to the supernatant to a final concentration of 20%, and incubated at 4°C for overnight. Precipitated proteins were removed by centrifugation at 12,000 × g and 4°C for 60 min. Then 2 volumes of absolute ethanol was added to the supernatant, and incubated at 4°C for 2 h. EPS was pelleted by centrifugation at 12,000 × g and 4°C for 10 min. The EPS was air dried and dissolved in H₂O. Then, the EPS was subjected to ion-exchange chromatography using a HiTrap DEAE column (GE healthcare) with linear gradient elution. Fractions containing EPS were merged and concentrated by ultrafiltration, and subsequently subjected to size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (GE healthcare).

AGP was isolated from plasma samples using a two-step ion exchange chromatography method according to Asao et al. [18 (link)]. One mL plasma samples were applied to a HiTrap Desalting column (GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM citrate-phosphate buffer, pH 4. The desalted peak was applied to a HiTrap DEAE column (GE Healthcare) equilibrated with 20 mM citrate-phosphate buffer, pH 4. Fractions containing AGP were eluted with 20 mM citrate-phosphate buffer, pH 7, containing 200 mM NaCl, pooled and applied on two joined HiTrap Desalting columns equilibrated with 20 mM citrate-phosphate buffer, pH 4. The desalted peak was applied to a HiTrap SP column (GE Healthcare) equilibrated with 20 mM citrate-phosphate buffer, pH 4, and AGP was eluted with 20 mM citrate-phosphate buffer, pH 4.8. The eluted fractions were dialyzed against water and lyophilized.