His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drugscreening

Tryparedoxin peroxidase has recently been identified as a constituent of th e complex peroxidase system in the trypanosomatid Crithidia fasciculata [No goceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) Biol Chem 378. 827- 836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADP H by means of a cascade of three oxidoreductases: the flavoprotein trypanot hione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of th ese enzymes are presumed to be trypanocidal drugs. Here, we present the het erologous expression of a putative tryparedoxin peroxidase gene of Trypanos oma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (Tc H6TXNPx). The product was purified with a high yield (8.75 mg from 1 1 ferm entation broth of A(600) 2.1) from the cytosolic fraction of sonified Esche richia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatograph y. TcH6TXNPx proved to be fully active when tested with heterologous trypar edoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx display ed ping-pong kinetics with a k(cat) of 1.7 s(-1) and limiting K-m values of 51.8 mu M and 1.7 mu M for t-butyl hydroperoxide and CfTXN2H6, respectivel y.