Cloning, expression and purification of Trypanosoma cruzi thioredoxin

PIATTONI, CV; BLANCATO, VS; MIGLIETTA, H; IGLESIAS, AA; GUERRERO, SA

Rosario, Argentina

Congreso; XX Reunión de la Sociedad Argentina de Protozoología; 2004

Sociedad Argentina de Protozoología

All forms of life have developed enzymatic systems to detoxify reactive oxygen species (ROS). The thioredoxin (TRX)-thioredoxin reductase (TRXR) system plays an important role in oxidative stress response in Archea, Eubacteria and Eukarya. In addition, TRX-TRXR system is involved in a variety of cellular redox reactions, DNA synthesis and transcription regulation, cell growth and apoptosis. Thioredoxins are small proteins with a molecular mass of 12 kDa. A redox active disulfide WCGPC motif is highly conserved amongst TRX’s family. In this work we report the cloning of a gene encoding TRX in Trypanosoma cruzi (TcTRX), and heterologous expression followed by purification of the recombinant protein. The results obtained by RT-PCR shows the presence of the mRNA of this gene. By southern blot analysis we demonstrated that the gene encoding TRX in T. cruzi is single copy. We used the pRSET A expression vector for trx sub-cloning and Escherichia coli BL21 (DE3) cells to produce the fusion protein. The recombinant six-histidine tagged TRX was purified by affinity metal chromatography. The deduced amino acid sequence of this protein has similarity to previously characterized Trypanosoma brucei brucei and Leishmania major TRXs. The purified TcTRX proved to be biologically active using an insulin reduction assay. By western blotting TcTRX was recognized by antibodies raised against recombinant MBP-T. brucei brucei TRX. Epimastigotes of T. cruzi display strong immunoreactivity to the antibodies raised against MBP-TbbTRX, thus suggesting the occurrence of TRX in this organism.