MOLECULAR AND IMMUNOLOGICAL CHARACTERIZATION OF A TRYPANOSOMA-CRUZI PROTEIN HOMOLOGOUS TO MAMMALIAN ELONGATION-FACTOR-1-GAMMA

In previous studies, we reported the characterization of three Trypano soma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP). U sing antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins. Comparison of the two sequences with amino acid sequ ences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1 beta . Thus, the proteins were named TcEF-1 beta 25 and TcEF-1 beta 30. In the present study we used a double immunoscreening strategy that allow ed us to isolate a cDNA clone corresponding to the 45 kDa protein. The protein sequence revealed 31% identity and 61% homology with human an d Artemia salina EF1 gamma and therefore was named TcEF-1 gamma Moreov er, three putative phosphorylation sites at position 51 (CSPC), at pos ition 90 (RTPL) and at position 265 (PSPF) were found in the TcEF-1 ga mma sequence. These sites are compatible with the notion that TcEF-1 g amma could be the target of phosphorylation by protein kinase(s). Rand om primed cDNA hybridized with a single 1.4 kb mRNA found in epimastig ote, trypomastigote and amastigote forms. In addition, Southern blot a nalysis of genomic DNA suggested that the protein is encoded by a sing le gene. The TcEF-1 gamma cDNA was subcloned into the pGEX-4T-3 vector for expression in Escherichia coli. Antibodies against the fusion pep tide allowed us to identify the weight sizes of the native protein (48 kDa) and its major degradation product (24.4 kDa) which are in close agreement with those of EF1-gamma from Artemia salina and Schizosaccha romyces pombe. These antibodies reacted against macrophage cell line J 774 extracts which indicates that EF-1 gamma epitopes were conserved t hroughout evolution.