Escherichia coli 987P pilus: Purification and partial characterization

R. E. Isaacson, P. Richter

Research output: Contribution to journalArticle

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Abstract

The E. coli somatic pilus, 987P, has been purified after removal by homogenization from a 987P+ enterotoxigenic E. coli. Cell-free pili were precipitated by the addition of MgCl2, collected, and dissolved in MgCl2-free buffer. Five cycles of precipitation and dissolving resulted in a preparation of 987P that was judged to be homogeneous based on electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the electron microscope, 987P was rod shaped, having a diameter of 7 nm and an apparent axial hole. Cells and membrane vesicles were not observed in the purified pilus preparation. Electrophoresis of 987P through sodium dodecyl sulfate-polyacrylamide gels resulted in a single band when the sample was denatured in the absence of mercaptoethanol and in two bands when the sample was denatured in the presence of mercaptoethanol. The calculated molecular weight of 987 was variable, depending upon the polyacrylamide concentration and whether mercaptoethanol was included in the denaturing solution. Chemically, 987P is composed primarily of protein but also contains an unidentified amino sugar. The amino terminal amino acid of 987P is alanine and its isoelectric point is pH 3.7. 987P possesses no detectable hemagglutinating activity.

Original languageEnglish (US)
Pages (from-to)784-789
Number of pages6
JournalJournal of Bacteriology
Volume146
Issue number2
StatePublished - Jan 1 1981

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Mercaptoethanol
Magnesium Chloride
Escherichia coli
Sodium Dodecyl Sulfate
Amino Sugars
Enterotoxigenic Escherichia coli
Isoelectric Point
Alanine
Electrophoresis
Polyacrylamide Gel Electrophoresis
Electron Microscopy
Buffers
Molecular Weight
Cell Membrane
Electrons
Amino Acids
Proteins

Cite this

Escherichia coli 987P pilus : Purification and partial characterization. / Isaacson, R. E.; Richter, P.

In: Journal of Bacteriology, Vol. 146, No. 2, 01.01.1981, p. 784-789.

Research output: Contribution to journalArticle

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AB - The E. coli somatic pilus, 987P, has been purified after removal by homogenization from a 987P+ enterotoxigenic E. coli. Cell-free pili were precipitated by the addition of MgCl2, collected, and dissolved in MgCl2-free buffer. Five cycles of precipitation and dissolving resulted in a preparation of 987P that was judged to be homogeneous based on electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the electron microscope, 987P was rod shaped, having a diameter of 7 nm and an apparent axial hole. Cells and membrane vesicles were not observed in the purified pilus preparation. Electrophoresis of 987P through sodium dodecyl sulfate-polyacrylamide gels resulted in a single band when the sample was denatured in the absence of mercaptoethanol and in two bands when the sample was denatured in the presence of mercaptoethanol. The calculated molecular weight of 987 was variable, depending upon the polyacrylamide concentration and whether mercaptoethanol was included in the denaturing solution. Chemically, 987P is composed primarily of protein but also contains an unidentified amino sugar. The amino terminal amino acid of 987P is alanine and its isoelectric point is pH 3.7. 987P possesses no detectable hemagglutinating activity.

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