[Purification of E. coli invasin IbeA-binding protein in intestinal epithelial cells]

Nan Fang Yi Ke Da Xue Xue Bao. 2009 Dec;29(12):2375-8.
[Article in Chinese]

Abstract

Objective: To purify IbeA-binding protein from intestinal epithelial Caco-2 cells.

Methods: Recombinant IbeA was purified, and 1, 5, and 10 microg/ml His-IbeA and bovine serum albumin (control) were preincubated with confluent Caco-2 monolayer for 30 min at 4degrees celsius;. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu(2+) sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay.

Results: E44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0.

Conclusion: Two novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caco-2 Cells
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism*
  • Epithelial Cells / microbiology
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification*
  • Escherichia coli Proteins / metabolism
  • Humans
  • Intestinal Mucosa / metabolism
  • Intestines / cytology
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • CusC protein, E coli
  • Escherichia coli Proteins
  • Membrane Proteins
  • Recombinant Proteins