Expression in insect cells of active mature cruzipain from T. cruzi, containing its C-terminal domain.

VANINA ALVAREZ; FABIOLA PARUSSINI; LENA ÅSLUND; JUAN JOSÉ CAZZULO

Villa Carlos Paz, Córdoba

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. SAIB 2002; 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. SAIB.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the C1 family, which contains, as the mature enzyme, a C-terminal domain 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. Therefore, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule (cruzainDc) has been determined. We have been able to express active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 2 mg per liter 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 C-terminal domain, and has kinetic properties similar to thos of native cruzipain, with some differences in substrate specificity. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.