Isolation and Characterization of the α -sialyl-β -2,3-galactosyl-Specific Adhesin from Fimbriated Escherichia coli
The α -sialyl-β -2,3-galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-fimbrial adhesin complex. A crude cell extract was partially dissociated into fimbriae and an adhesin-enriched fraction by heating to 70 degrees C. From...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 10; pp. 3462 - 3466 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Washington, DC
National Academy of Sciences of the United States of America
01.05.1987
National Acad Sciences |
Subjects | |
Online Access | Get full text |
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Summary: | The α -sialyl-β -2,3-galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-fimbrial adhesin complex. A crude cell extract was partially dissociated into fimbriae and an adhesin-enriched fraction by heating to 70 degrees C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO4/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of Mr≈ 106that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino acid compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same specificity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal anti-adhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.84.10.3462 |