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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 (NH?)-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.

SUBMITTER: Tanaka T

PROVIDER: S-EPMC3014551 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Expression, identification and purification of Dictyostelium acetoacetyl-coa thiolase expressed in Escherichia coli.

Tanaka Takeshi T Shima Yasuyuki Y Ogawa Naoki N Nagayama Koki K Yoshida Takashi T Ohmachi Tetsuo T

International journal of biological sciences 20101230 1

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 ...[more]

PMID: 21209787

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Project description:BackgroundClonorchis sinensis (C. sinensis) inhabits in bile duct of the host. However, the mechanisms involved in why C. sinensis can survive in the bile environment containing lipids have not yet been explored. In this study, C. sinensis acetoacetyl-CoA thiolase (CsACAT), a member of the thiolase family which has a key role in the beta oxidation pathway of fatty acid production, was identified and characterized to understand its potential role in adapting to the bile environment.MethodsThe encoding sequence, conserved domains and spatial structure of CsACAT were identified and analyzed by bioinformatic tools. Recombinant CsACAT (rCsACAT) was obtained using a procaryotic expression system. The expression pattern of CsACAT was confirmed by quantitative real-time PCR, western blotting, and immunofluorescence. Gradients of lecithin were then set to culture C. sinensis adults in vitro and the survival rate of C. sinensis was analyzed, as well as the expression level and enzymatic activity of CsACAT in different lipid environments. Hypercholesteremia rabbit models were established by feeding with a hyperlipidemic diet and then infected intragastrically with C. sinensis. One and a half months later, the worm burdens and the expression level of CsACAT was detected.ResultsCsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis. CsACAT was specifically localized at the vitellarium and sub-tegumental muscle layer in adult worms. The mRNA level of CsACAT in eggs was higher than those in adult worms and metacercariae. When adult worms were cultured with higher concentration of lecithin, the expression level and enzyme activity of CsACAT were up-regulated. The survival rate of adult worms was higher than control group. More adult worms were recovered from hypercholesteremia rabbit models. The expression level of CsACAT in these worms was higher than control group.ConclusionsOur results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels. C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.

Project description:The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.

Dataset Information

Expression, identification and purification of Dictyostelium acetoacetyl-coa thiolase expressed in Escherichia coli.

Publications

Expression, identification and purification of Dictyostelium acetoacetyl-coa thiolase expressed in Escherichia coli.

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