cAMP receptor protein from Trypanosoma cruzi: purification and cloning of a short sequence of the corresponding cDNA

RESUMO

cAMP is involved in the differentiation of Trypanosoma cruzi, the causative agent of Chagas' disease. cAMP levels are elevated in the infective, non-dividing metacyclic trypomastigote stage, with respect to the non-infective, proliferative, epimastigote stage. In both stages three is a cAMP receptor protein (CARPT), with unique properties that differentiate it from the regulatory subunits of the cAMP-dependent protein kinase (RI and RII). The CARPT from T. cruzi epimastigotes was purified using ion-exchange chromatography, affinity chromatography and gel filtration. After the final step of purification, two protein bands were obtained, p89 and p70, corresponding to the intact CARPT and its proteolytic product. These two CARPT polypeptides were utilized to prepare polyclonal antibodies in rabbits. Previous results from our laboratory showed that CARPT cross-reacts with polyclonal antibodies prepared against the regulatory subunit (RII) of the cAMP-dependent protein kinase (PKA). As expected from these results, the anti-CARPT antibody recognized purified RII protein in an ELISA assay. The anti-CARPT antibodies were used for immunoblot analyses of epimastigote lysates. The two bands corresponding to the CARPT (p89 and p70), as well as a p40 band, were recognized. Immunoscreening of a T. cruzi lambda ZAP cDNA library with these anti-CARPT polyclonal antibodies yielded one positive clone (pBSCARPT) which contained a 540 bp insert. Northern analyses using the pBSCARPT clone as a probe, showed a 5.2 kb mRNA band in epimastigotes, which were grown in culture from 2 to 10 days in LIT medium. Sequence analyses of the 540 bp insert have failed to show homology to other gene sequences in the database.(ABSTRACT TRUNCATED AT 250 WORDS)