title: 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 50,502Da 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.
authors.affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Gopal
authors.initials: G
authors.lastname: Kulkarni
authors.forename: Nirupama A
authors.initials: NA
authors.lastname: Sabnis
authors.forename: Ben G
authors.initials: BG
authors.lastname: Harris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005649:Fumarate Hydratase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D005649:Fumarate Hydratase