A Chromatographic Method for the Purification of K99 Pili from Enterotoxigenic Escherichia coli

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Abstract
Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (wt/vol) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0.02 M Tris/HCl, pH 8.6, containing 0.05 M NaCl and DEAE-Sephadex A-50 equilibrated with 0.05 M phosphate buffer, pH 7.2. The purified pili yielded a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated MW of 13,000. Attempts to purify pili by other methods (MgCl2 precipitation and chromatofocusing) were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously, the N-terminal amino acid was apparently blocked.