Cloning, expression, and purification of fumarase from the parasitic nematode Ascaris suum

Abstract

The cDNA encoding fumarase, an enzyme catalyzing reversible hydration of fumarate to l-malate, from the parasitic roundworm Ascaris suum, has been cloned, sequenced, over-expressed in Escherichia coli, and purified. The single open reading frame translates into a protein of 50,502 Da containing 467 amino acids. It shows 82, 77, and 58% identity with Caenorhabditis elegans, human, and E. coli fumC fumarases, respectively. The A. suum fumarase shows the signature sequence motif (GSSIMPGKVNPTQCE), which defines not only the class II fumarase family but also a much broader superfamily of proteins containing GSSxMPxKxNPxxxE motif. The coding region was cloned into pET101D-directional TOPO expression vector and transformed into E. coli BL21 Star (DE3). The protein after induction was expressed at high levels, almost 10% of the soluble protein, purified to near homogeneity, and appears identical to the enzyme purified from Ascaris suum.