Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis
- PMID: 24739240
- PMCID: PMC4143643
- DOI: 10.1292/jvms.13-0623
Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis
Abstract
The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme has been characterized in other species, but not in the genus Babesia, which causes major losses in the livestock industries worldwide. Therefore, we isolated, cloned and expressed the wild-type B. bovis dxs cDNA in Escherichia coli and evaluated its enzymatic activity in vitro. DNA sequence analysis revealed an open reading frame of 2061 bp capable of encoding a polypeptide of 686 amino acid residues with a calculated isoelectric point of pH 6.93 and a molecular mass of 75 kDa. The expressed soluble recombinant fusion DXS protein was approximately 78 kDa, which is similar to the native enzyme identified from the parasite merozoite using anti-rDXS serum. The recombinant fusion DXS enzyme exhibited Km values of 380 ± 46 µM and 790 ± 52 µM for D,L-glyceraldehyde 3-phosphate and pyruvate, respectively. In this work, we present the first cloning, expression and characterization of DXS enzyme from B. bovis.
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