Abstract: The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins bind to specific cell surface receptors. To confirm binding specificity, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. The high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning. Antibody fragments were isolated from a naive single-chain Fv (scFv) library biopanned against PA83. Four scFv proteins were found to bind to PA83, the best one exhibiting a 10 nM Kd. Two scFv proteins, scFv #1 and scFv #4, had similar affinities for PA32 and PA83, confirming the recombinant fragment was folded correctly.

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.

Abstract: A method for making recombinant fluorescent protein standard particles for calibration of fluorescence instruments.