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Observation of the Intracellular Behavior of Recombinant Yersinia pseudotuberculosis Invasin Protein
Author(s) -
Koga Hisashi,
Izawa Ichiro,
Araki Norie,
Saya Hideyuki,
Mimori Tatsuyuki
Publication year - 2005
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2005.tb03729.x
Subject(s) - yersinia pseudotuberculosis , yersinia , biology , biotinylation , intracellular , microbiology and biotechnology , recombinant dna , cytoplasm , bacterial outer membrane , bacteria , biochemistry , escherichia coli , virulence , genetics , gene
In this study, we observed the intracellular behavior of recombinant invasin, a 103‐kDa outer membrane protein of Yersinia pseudotuberculosis . To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST‐fused invasin C‐terminal portion protein (GST‐INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin‐mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, 125 I‐uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA‐insoluble (polymerized‐actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial‐free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.