A chromatographic method for the purification of K99 pili from enterotoxigenic Escherichia coli.
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% (w/v) 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 SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. 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.
D000596:Amino Acids / Q000032:analysis; D000942:Antigens, Bacterial / Q000302:isolation & purification; D002458:Cell Fractionation / Q000379:methods; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000096:biosynthesis; D004926:Escherichia coli / Q000276:immunology / Q000648:ultrastructure*; D010861:Fimbriae, Bacterial / Q000276:immunology; D007122:Immunoelectrophoresis
D000596:Amino Acids; D000942:Antigens, Bacterial; D004768:Enterotoxins