Method for screening inhibitors of the toxicity of Bacillus anthracis

US6329156A · kind A · utility

Assignee

• The Regents of the University of California · US

Inventors

• Nick M. Cirino · Cleveland Heights, US
• Paul J. Jackson · London, GB
• Bruce E. Lehnert · Los Alamos, US

Key dates

• Filing date: Mar 22, 1999
• Grant date: Dec 11, 2001
• Expiry date: Mar 22, 2019

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/18
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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.