RESUMEN
Trypanosomatids are unicellular organisms that colonize a wide diversity of environments and hosts. For instance, Trypanosoma cruzi is a human pathogen responsible for Chagas diseases, while Leishmania tarentolae infects amphibians and became a biotechnological tool suitable for recombinant protein expression. T. cruzi antigens are needed for the development of improved epitope-based methods for diagnosis and treatment of Chagas disease. Molecular cloning for the production of recombinant proteins offers the possibility to obtain T. cruzi antigens at high yield and purity. L. tarentolae appears as the ideal expression host to obtain recombinant T. cruzi antigens with a structure and posttranslational modifications typical of trypanosomatids. In this chapter, we present a protocol for the analytical to mid-scale production of recombinant T. cruzi antigens, using L. tarentolae as expression host (LEXSY® inducible system).
Asunto(s)
Antígenos de Protozoos/genética , Clonación Molecular/métodos , Leishmania/genética , Trypanosoma cruzi/genética , Enfermedad de Chagas/parasitología , Vectores Genéticos/genética , Humanos , Plásmidos/genética , Proteínas Recombinantes/genética , Transfección/métodosRESUMEN
Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.
