Apply the concentrated component I to a column of Sephacryl S-300 (2.5x95cm) equilibrated with 50 mM PB, pH 7.5, and elute with the buffer. Collect the fractions containing component I and concentrate by ultrafiltration as described above. Check the purity of the component using SDS-PAGE. If the preparation contains any impurities, run repeat if preparation the gel filtration again on the column of Sephacryl S-300 under the contains impurities same conditions as above.
By these purification procedures, l-3mg of component I and 20-40 mg of component II are recovered from 4 I of culture of C. botulinum type C strain 92-13. The mixture of untrypsinized component I and trypsinized component II has high lethal activity in mice (specific activity is about 2xl04 mouse intraperitoneal 50% lethal doses per mg of protein), although each of these components alone shows very low activity even after trypsinization. The purified components I and II each show one band in SDS-PAGE, and their molecular weights as determined by the electrophoresis are 45kDa and 100 kDa, respectively.
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