A METHOD FOR THE TARGETING OF PROTEINS PRODUCED BY YERSINIA INTO THE CYTOSOL OF EUKARYOTIC CELLS

摘要

A method of delivering a polypeptide to eukaryotic cells based on the YopE system of Yersinia involves one step translocation of the polypeptide in the response to targeting signals provided by the first 100 amino acid residues of the YopE protein of the first 100 amino acid residues of the YopE protein of Yersinia. This method requires functional SycE chaperone activity. The translocated polypeptide is delivered to the cytosol of the eukaryotic cells. In general, this method comprises: (1) providing a nucleic acid segment encoding a fusion protein, the fusion protein including: (a) the composite YopE targeting signal; and (b) a polypeptide to be translocated, the nucleic acid segment capable of expression in Yersinia cells; (2) expressing the nucleic acid segment in Yersinia cells; and (3) contacting Yersinia cells expressing the nucleic acid segment which eukaryotic cells in the presence of functional SycE chaperone activity so that the fusion protein including the polypeptide is translocated to the cytosol of the eukaryotic cells. The method is not limited to Yersinia cells, but can be used with other Gram-negative bacteria as well. The present invention also includes screening methods and methods of blocking the introduction of a bacterial protein translocated by a translocation system in a Gram-negative bacterium employing a targeting signal into a eukaryotic cell.