title: Expression, identification and purification of Dictyostelium acetoacetyl-coa thiolase expressed in Escherichia coli.
abstract: Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tanaka
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Shima
authors.forename: Naoki
authors.initials: N
authors.lastname: Ogawa
authors.forename: Koki
authors.initials: K
authors.lastname: Nagayama
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Ohmachi
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D019976:Cloning, Organism; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D000101:Acetyl-CoA C-Acetyltransferase