Observation of the Intracellular Behavior of Recombinant Yersinia pseudotuberculosis Invasin Protein

Abstract

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, 125I‐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.

Tissular model/sensor seamless system for qualified analysis and its characterization

“…The blots were incubated with the first antibody (eNOS, mAChR, vWF, -Actin and GAPDH antibody) at a dilution of 1:250 for 1 h at room temperature, extensively washed, and incubated with the second antibody (horseradish peroxidase-conjugated antimouse immunoglobulin antibody) at a dilution of 1:500 for one more hour. The proteins were visualized on a Duo Lux (Pierce) [21][22][23].…”