title: Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum.
abstract: The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an overall yield of 51%. The bacterially expressed enzyme exhibits kinetic constants identical to those measured for native A. suum NAD-malic enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
authors.forename: L
authors.initials: L
authors.lastname: Chooback
authors.forename: W E
authors.initials: WE
authors.lastname: Karsten
authors.forename: G
authors.initials: G
authors.lastname: Kulkarni
authors.forename: S R
authors.initials: SR
authors.lastname: Nalabolu
authors.forename: B G
authors.initials: BG
authors.lastname: Harris
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D000818:Animals; D017165:Ascaris suum / Q000201:enzymology* / Q000235:genetics; D002853:Chromatography, Liquid; D004926:Escherichia coli; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)