New Method for isolation of immunologically pure pili from Escherichia coli.
A new technique for purification of bacterial pili was developed and applied to Escherichia coli strains isolated from the urine of patients with symptomatic urinary tract infections. After mechanical detachment from the bacterial cells, the pili were concentrated by precipitation with ammonium sulfate, dialyzed, and solubilized in buffer containing deoxycholate. The fraction containing the pili was purufied further by ultracentrifugation in a sucrose gradient and by elution through a Sepharose 4B column in 6 M urea buffer. The pilus filaments were not dissociated by concentrated urea and were eluted in the void volume of the column. The purified pili had a molecular weight of 17,000. The isoelectric point of the pili from one of the strains was 4.9, and about 43% of the amino acids were hydrophobic. Hyperimmunesera raised in rabbits against the purified pili did not contain detectable antibodies to the lipopolysaccharide O antigen or to the capsular polysaccharide K antigen of the homologous strain. The pili obtained by this purification procedure are free from other detectable bacterial surface antigens, and the purified pilus filaments are of relatively homogeneous size. This procedure enables purification of the pili also from flagellated strains.
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000032:analysis; D001431:Bacteriological Techniques; D002458:Cell Fractionation / Q000379:methods*; D002847:Chromatography, Agarose; D004926:Escherichia coli / Q000648:ultrastructure*; D010861:Fimbriae, Bacterial / Q000032:analysis / Q000276:immunology; D007526:Isoelectric Point; D008970:Molecular Weight; D011817:Rabbits; D014461:Ultracentrifugation
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins