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Title: Method for screening inhibitors of the toxicity of Bacillus anthracis

Abstract

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

Inventors:
 [1];  [1];  [1]
  1. (Los Alamos, NM)
Issue Date:
Research Org.:
University of California; Los Alamos National Laboratory (LANL), Los Alamos, NM
Sponsoring Org.:
USDOE
OSTI Identifier:
874161
Patent Number(s):
6329156
Application Number:
09/273,839
Assignee:
The Regents of the University of California (Los Alamos, NM) LANL
DOE Contract Number:  
W-7405-ENG-36
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
method; screening; inhibitors; toxicity; bacillus; anthracis; protective; antigen; integral; mechanism; anthrax; poisoning; cloning; expression; purification; 32; kda; fragment; pa32; described; expressed; fusion; construct; stabilized; green; fluorescent; protein; egfp-pa32; proteins; capable; binding; specific; cell; surface; receptors; determined; microscopy; flow; cytometric; assay; confirm; specificity; non-fluorescent; pa83; competitively; inhibit; employed; rapid; screen; compounds; disrupts; cells; additionally; intracellular; levels; ease; recombinant; attractive; vaccine; candidate; therapeutic; treatment; /435/436/

Citation Formats

Cirino, Nick M., Jackson, Paul J., and Lehnert, Bruce E. Method for screening inhibitors of the toxicity of Bacillus anthracis. United States: N. p., 2001. Web.
Cirino, Nick M., Jackson, Paul J., & Lehnert, Bruce E. Method for screening inhibitors of the toxicity of Bacillus anthracis. United States.
Cirino, Nick M., Jackson, Paul J., and Lehnert, Bruce E. Mon . "Method for screening inhibitors of the toxicity of Bacillus anthracis". United States. https://www.osti.gov/servlets/purl/874161.
@article{osti_874161,
title = {Method for screening inhibitors of the toxicity of Bacillus anthracis},
author = {Cirino, Nick M. and Jackson, Paul J. and Lehnert, Bruce E.},
abstractNote = {The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2001},
month = {1}
}

Patent:

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