Heterologus expression of trypanothione reductase from Leishmania donovani in a prokaryotic system

A process for heterologus expression of functionally active enzyme trypanothione reductase of Leishmania donovani strain Dd8 (WHO Reference No. MHOM/IN/80/Dd8;

• available in ATCC, No. 50212) in a prokaryotic system, said process comprising the steps of:
• (a) amplifying, identifying and isolating the Open Reading Frame (ORF) of trypanothione reductase having the nucleotide sequence of SEQ ID NO:
• 1 and the corresponding protein of SEQ ID NO:
• 2 using forward primer consisting of SEQ ID NO:
• 5 and reverse primer consisting of SEQ ID NO:
• 6,
• (b) cloning of the amplified product of SEQ ID NO:
• 1 of step (a) in commercial TA cloning vector,
• (c) transforming and propagating the clone of step (b) in E. coli cells,
• (d) purifying plasmid DNA of step (c) and using it as a template for amplification of LddTR ORF having Nco1 and Bam H1 flanking restriction sites for directional cloning in an expression vector,
• (e) subcloning of LddTR-ORF in commercial pET41 expression vector,
• (f) transforming the cloned LddTR-ORF of step (e) in E. coli cells and studying the expression of LddTR protein in bacterial lysate under different conditions of expression and selection of conditions for maximum expression,
• (g) purifying the LddTR protein of SEQ ID NO:
• 2 from step (f) to homogeneity, and
• (h) analyzing biochemical parameters (substrate specificity, specific activity, Kin, Vmax, and effect of inhibitors) of the expressed trypanothione reductase enzyme (LddTR) to establish the heterologous expression of trypanothione reductase of Leishmania donovani of SEQ ID NO:
• 2 in a prokaryotic system.