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


CONCLUSIONS
Two novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.
Hui
Chang-ye
CY
Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. hcy_sypu@163.com
Guo
Yan
Y
Li
Jun
J
Hao
Xiao-yan
XY
Cao
Hong
H
Huang
Sheng-he
SH
D018938:Caco-2 Cells; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
C096350:CusC protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins