Bacillus Anthracis, Lethal Factor, Recombinant (Anthrax)

Blue Ice

90

Supplied as a lyophilized powder in 5mM HEPES, 50mM sodium chloride, pH 7.5. Reconstitute with 100ul sterile ddH2O. Handle Gently. Do not vortex.

Bacillus anthracis is the etiological agent of anthrax. Major virulence factors produced by Bacillus anthracis are the gamma-linked, poly-D-glutamic acid capsule and an exotoxin composed of three components, protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are not toxic separately but act in binary combinations. The complex of PA, the cell binding component, with LF, one of the enzymatic moieties, is termed “lethal toxin” and can cause death. PA and the enzymatic EF together cause skin edema. After secretion, PA is cleaved by membrane peptidases, allowing the 63kD carboxy terminal fragment to oligomerize to a heptamer. Cleavage is an essential step in exposing the binding sites for EF and LF. The assembled PA complex and associated LF or EF enters the cell through endocytosis. PA mediates the transfer of LF and EF to the cytoplasm where these enzymes recognize and proteolyze their targets. The LF component, a zinc-dependent metalloprotease, cleaves the mitogen-activated protein kinase kinase (MAPKK), impairing its function and thereby blocking the MAPK signaling pathway. EF is a calmodulin-dependent adenylate cyclase which creates in the cell excess amounts of cyclic-AMP, resulting in edema. Current research is focused on the development of an effective vaccine as well as screening for potent inhibitors of the LF enzymatic activity.

Source:
Recombinant protein corresponding to Bacillus anthracis Lethal Factor.

Molecular Weight:
~90kD

Biological Activity:
Assessed for specific activity in units/mg protein, a synthetic peptide containing a single cleavage site for LF. Assessed for cytotoxicity in the presence of 1ug/ml PA using J774A1 cells. When J774A.1 cells are treated with LF alone, no toxicity is seen at 1ug/ml (12nM). The effective concentration 50% (ECso) of this LF lot meets specifications.

Specific Activity:
~0.310units/mg. This activity is 1.4 times the activity of the control lot of lethal factor.

Unit Definintion:
One unit is defined as the amount of lethal factor needed to catalyze the release of 1.0umole of cleaved MAPKKIde/minute at 37°C in 20mM HEPES, pH 8.2.

Endotoxin:
7.5EU/mg (Kinetic chromagenic LAL assay)

Tissue Culture Application:
For tissue culture applications, medium containing glutamine must be fresh. Ammonium ion is released when glutamine breaks down, and may prevent acidification of the endosome thereby inhibiting translocation of LF or EF into the cytosol. A stable form of glutamine may
be used.

Storage and Stability:
Lyophilized powder may be stored at -20°C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.