Deae sepharose fast flow

M. sporium strain 5 [American Type Culture Collection (ATCC) 35069] was cultured in nitrate mineral salt media (ATCC 1306) at 30°C until optical density at 655 nm reached 8 to 10 with a methane:air (v/v) ratio of 10 to 15%. The cells were harvested by centrifugation (11,300g) for 20 min at 4°C. The cell pellets were suspended in a lysis buffer containing Mops (25 mM), NaCl (25 mM), sodium thioglycolate (8 mM), l-cysteine (2 mM), (NH₄)₂Fe(SO₄)₂·6H₂O (200 μM), MgCl₂ (5 mM), deoxyribonuclease (DNase) (0.25 μl/ml), and phenylmethylsulfonyl fluoride (PMSF, 0.04 mg/ml) at pH 6.5. The cell suspension was sonicated at 4°C (CV334 model, Sonics), and the lysate was centrifuged at 30,000g for 45 min at 4°C. The supernatant was carefully decanted and filtered through a 0.22-μm membrane (Merck Millipore). The filtrate was loaded onto DEAE Sepharose Fast Flow, Superdex 200, and Q Sepharose Fast Flow columns attached to the ÄKTA Pure 25 L fast protein liquid chromatography system (GE Healthcare) to purify MMOH with >95% purity (30 (link)). The purified MMOH was applied to a ferrozine assay to monitor the iron-ferrozine complex (562 nm), and the results revealed 3.9 to 4.2 Fe/MMOH with coefficient of determination (R²) values >0.999 (14 (link)).

IgM-secreting hybridoma cells (3F3F5 clone) were applied to the Hybridoma-SFM (Thermo Fisher Scientific Inc.), and grown at 125 rpm in a shaker incubator containing 8% CO₂ at 37°C. After 5 days, cell culture supernatants were obtained by centrifugation at 3,000 rpm for 10 min, and purified using DEAE Sepharose^™ Fast Flow (GE Healthcare Co.) and HiPrep^™ 16/60 Sephacryl^™ S-300 HR (Ge Healthcare Co.). The samples were loaded onto a SDS-PAGE, analyzed by Coomassie blue staining and Western blotting (28 (link)).

Starter culture was prepared as previously described of inoculum, then batch fermentation was performed in a 5.0L capacity fermentor (Biotech-2002; Bao Xing Bio-Engineering Equipment Co., Ltd, Shanghai, China) at 37°C with an inoculum concentration of 3.0% to obtain the fermented broth (15 (link)). Extracellular polymeric substances were obtained as described previously (34 (link)). The crude EPS fractions were separated and purified according to procedures described by Lin et al with slight modifications (39 (link)). The obtained crude EPS fractions were then lyophilized in a freeze-drier (FD-1C-50; Boyikang, Beijing, China). The acidic EPS was purified by using anion exchange chromatography on a DEAE Sepharose Fast-Flow (GE Healthcare, Chicago, IL, USA) column (1.6×20 cm) with the NaCl gradient (0~1M) as the elution buffer at a flow rate of 1.0 ml/min. The purified acidic EPS was collected by using a fraction collector with 5 ml per tube. The eluent was assayed for carbohydrate contents by the phenol-sulfuric acid method described by DuBois et al (40 (link)). The peak fractions containing polysaccharides were pooled and dialyzed with deionized water every 6 h for 48 h and freeze-dried.