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The major cysteine protease of Trypanosoma cruzi, cruzain, has been previously
expressed in Escherichia coli as a fusion polypeptide. The proteolytic
processing events required to obtain active, mature cruzain from the recombinant
expression system have been characterized using mutational analysis of the
cloned gene. An inactive variant of cruzain (cruzain-C25A) revealed that the
proteolytic cleavage of the COOH-terminal domain from the recombinant cruzain is
independent of cruzain activity. This cleavage event, presumably performed by
another protease, was reduced, although not completely eliminated, in a variant
in which the cleavage recognition site was altered (cruzain-E219P). To obtain a
homogeneous COOH terminus of the recombinant enzyme, a truncated form of cruzain
(cruzain-delta c) was engineered by insertion of a stop codon in the gene at a
site corresponding to autoproteolysis observed with the native enzyme, purified
from epimastigotes. Diffraction quality crystals of recombinant cruzain
(cruzain) and the truncated variant (cruzain-delta c) have been produced and
characterized. Cruzain and cruzain-delta c were cocrystallized with the peptide
fluoromethyl ketone (FMK) inhibitors, Z-Phe-Arg-FMK and Z-Phe-Ala-FMK,
respectively, (where Z is benzyloxycarbonyl). The crystals are monoclinic, space
group P2(1), with a = 45.5 A, b = 51.0 A, c = 45.7 A, and beta = 116.1 degrees.
One cruzain molecule is present in the asymmetric unit. The crystallographic
data reveal that the high resolution structure determination is feasible. This
system will facilitate the three-dimensional structure determinations and
biochemical analyses of cruzain and cruzain variants.
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