Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi.
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.
Eakin
A E
AE
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
McGrath
M E
ME
McKerrow
J H
JH
Fletterick
R J
RJ
Craik
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016054:DNA, Protozoan; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D007659:Ketones; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014961:X-Ray Diffraction
D016054:DNA, Protozoan; D007659:Ketones; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi