Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9894299 | Protein Expression and Purification | 2005 | 8 Pages |
Abstract
Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are Km, 50 μM; kcat, 18,181 minâ1; and kcat/Km, 6.06 Ã 106 Mâ1 sâ1. The yield of recombinant LdTR was â¼16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.
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Authors
Mukul K. Mittal, Smita Misra, Mohammad Owais, Neena Goyal,