Expression in insect cells of active mature cruzipain from Trypanosoma cruzi, containing its C-terminal domain.
Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.
Alvarez
Vanina
V
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martin, 1650 San Martin, Provincia de Buenos Aires, Argentina.
Parussini
Fabiola
F
Aslund
Lena
L
Cazzulo
Juan J
JJ
D000818:Animals; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C063214:cruzipain