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J. Biol. Chem., Vol. 266, Issue 30, 20124-20130, Oct, 1991
CP Quinn, Y Singh, KR Klimpel and SH Leppla
Linker insertion mutagenesis was employed to create structural disruptions
of the lethal factor (LF) protein of anthrax toxin to map functional
domains. A dodecameric linker was inserted at 17 blunt end restriction
enzyme sites throughout the gene. Paired MluI restriction sites within the
linker allowed the inserts to be reduced from four to two amino acids.
Shuttle vectors containing the mutated genes were transformed into the
avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the
gene products. Mutations at five sites in the central one-third of the
sequence made the protein unstable, and purified protein could not be
obtained. Mutated LF proteins with insertions at the other sites were
purified and assessed for toxic activity in a macrophage lysis assay and
for their ability to bind to the protective antigen (PA) component of
anthrax toxin, the receptor binding moiety. Most insertions located in the
NH2-terminal one-third of the LF protein eliminated both toxicity and
binding to PA, while all four insertions in the COOH-terminal one-third of
the protein eliminated toxicity without affecting binding to PA. These data
support the hypothesis that the NH2-terminal domain contains the structures
required for binding to PA and the COOH-terminal domain contains the
putative catalytic domain of LF.
Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
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