Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli.

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Jayalakshmi R, Sumathy K, Balaram H

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli.

Protein Expr Purif. 2002 Jun;25(1):65-72.

PubMed ID

12071700 [ View in PubMed

]

Abstract

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Aspartic acid	Adenylosuccinate Synthetases (Protein Group)	Protein group	Humans	Unknown	Substrate	Details

Polypeptides

Name	UniProt ID
Adenylosuccinate synthetase	Q9U8D3	Details