Heterologus expression of trypanothione reductase from Leishmania donovani in a prokaryotic system
AssigneeCouncil of Scientific and Industrial Research
InventorsNeena Goyal, Mukul Mittal
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Abstract
The present invention relates to a process for heterologous expression and large-scale production of functionally active enzyme trypanothione reductase of Leishmania donovani in prokaryotic system.
Claims38
A process for heterologus expression of functionally active enzyme trypanothione reductase of Leishmania donovani strain Dd8 having accession ATCC 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 DNA SEQ ID No. I and corresponding protein SEQ ID No. 2 using forward primer having SEQ ID No. 3 and reverse primer having SEQ ID No. 4,
- (b) cloning of the amplified product of step (a) in cloning and expression vector,
- (c) subcloning of the amplified product of step (b) in cloning and expression vector,
- (d) transforming the cloned ORF of steps (b) & (c) in a prokaryotic heterologus cloning and expression system,
- (e) studying the expression of the ORF transformed in step (d) under different in vitro conditions, and
- (f) purifying the enzyme from step (d) to homogeneity, and
- (g) analyzing physical and biochemical properties of the enzyme to establish the heterologus expression of soluble native form of ORF Leishmania donovani strain Dd8 having accession ATCC 50212 in a prokaryotic system.
Oligonucelotide primers having SEQ ID No.3 (forward primer) and SEQ ID No.4 (Reverse primer) for isolating Open Reading Frame (ORF) of trypanothione reductase from Leishmania donovani strain Dd8 having accession ATCC 50212 and its heterologus expression in a prokaryotic system.
Cloning and expression vectors for isolating Open Reading Frame (ORF) of trypanothione reductase from Leishmania donovani strain Dd8 having accession ATCC 50212 and its heterologus expression in a prokaryotic system.
A cloning prokaryotic system for isolating and cloning Open Reading Frame (ORF) of trypanothione reductase from Leishmania donovani strain Dd8 having accession ATCC 50212 and its heterologus expression in a prokaryotic system.
A system of expression prokaryotic system for expressing Open Reading Frame of trypanothione reductase from Leishmania donovani strain Dd8 having accession ATCC 50212 and its heterologus expression in a prokaryotic system
Dependencies are inferred from the claim text.