Lysozyme size-exclusion chromatography

The initial PIA purification method was developed by Mack et al. (3). These authors used a different, two-step chromatography protocol involving size-exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose, and S-Sepharose. A similar purification method has been described recently to isolate a PIA-related polysaccharide polymer in E. coli (7). Briefly, E. coli cells were incubated in 50 raM Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and 0.1 M EDTA at room temperature for 2 h. Phenol/chloroform extraction steps were performed to separate protein and debris contamination from the polysaccharide. Samples were concentrated by ultrafiltration devices (10,000 MW cut off) and fractionated on a fast protein liquid chromatography (FPLC) system with a Sephacryl S-2000 column (equilibration and elution buffer 0.1 MPBS, pH 7.4). 

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