Development of a Protective Antigen capture system based on the N-terminal domain of Lethal Factor (Marie Curie funding awarded to Elzbieta Lis, 2013 – 2014)

Bacillus anthracis is a spore forming, Gram-positive bacterium that is the etiologic agent of Anthrax. A major virulence factor is the tripartite Anthrax toxin which is formed by three proteins Lethal factor, Edema factor and Protective antigen on the pX01 plasmid. Lethal factor (LF), a protease cleaving mitogen-activated protein kinase kinases, is comprised of four distinct domains, of which the N terminal domain (LF-D1) is responsible for binding to Protective antigen (PA), the factor responsible for translocating the toxin into the cell. In this study we determined the ability to attach histidine tagged, recombinantly expressed LF-D1, to the surface of an immobilized metal affinity chromatography resin (IMAC) via metal ions with a view to developing a system capable of removing PA from infected body fluid, such a system could then be used to treat the blood of patients with Anthrax. Figure 1 shows the theory behind the project.

Anthrax_conference_diagram
Figure 1: A diagram of the theory. PA in blood is passed through the IMAC column, where recombinant LF-D1 is bound. Due to the specificity between LF and PA binding, the PA is bound and therefore removed from the blood. Western blots and ELISAs for LF and PA were both performed before passing through the column and the elution from the column.

Recombinant LF-D1 was immobilized onto a series of IMAC resins containing one of the following metals; Ni2+, Co2+, Cu2+ and Zn2+ ions, to determine if one particular ion performed better than another. A series of experiments were performed to determine the PA concentration in simulated serum and blood, both before treatment through the rLF-D1 column and after.

Our results showed that rLF-D1 was unable to efficiently bind PA from simulated blood or serum, with differing metal ions having little effect on the efficiency of PA binding.