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Physical Characteristics of rLF and rPA: Effects on Enzymatic : Effects on Enzymatic Activity and Binding

Shine, N., Le, C., Eaton, L., Crawford, K.

Bacillus anthracis exotoxin is composed of three components; protective antigen (PA), lethal factor (LF), and edema factor (EF), which act in binary combinations. The complex of PA, the cell binding component, with LF, the metallo-endopeptidase moiety, is termed lethal toxin and can cause death. Recombinant forms of PA and LF are produced in an avirulent, non-capsulated, sporulation-suppressed B. anthracis host at List Labs. Electrospray Ionization mass spectrometry (ESI) analysis of several lots of LF indicate that truncated forms of LF are produced, as well as the full length. An HPLC protocol was used to analyze the percent full length toxin in each lot. The percent of truncated fragments in a given sample of LF was correlated to the enzymatic activity, which is assessed by measuring the specific activity of each lot using MAPKKideTM, a quenched fluorigenic peptide substrate for LF. An assay using PA63-coated plates was developed to study the effect of LF length on the binding of LF to PA. In order to determine whether addition of ZnCl2 would increase the enzymatic activity of LF, the kinetic parameters, Km and Vmax, were measured after incubation with several concentrations of ZnCl2. ESI of rPA also indicated that each lot contains a truncated form as well as full length. The stability of LF and PA at room temperature was assessed using HPLC. These studies are important for the assessment of characteristics of the proteins that may impact cytotoxicity.