Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria

被引:3
作者
Fillgrove, KL
Anderson, VE
Mizugaki, M
机构
[1] Case Western Reserve Univ, Dept Biochem, Cleveland, OH 44106 USA
[2] Tohoku Univ Hosp, Dept Pharmaceut Sci, Sendai, Miyagi, Japan
关键词
D O I
10.1006/prep.1999.1101
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The mitochondrial a,4-dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme for the beta-oxidation of unsaturated fatty acids. Import of this enzyme into the mitochondria requires a mitochondrial signal sequence at the amino terminus of the polypeptide chain which is processed/removed once inside the mitochondria, The cDNA of the full-length a,4-dienoyl-CoA reductase was previously cloned as pRDR181. PCR methodologies were used to subclone the gene encoding the functional a,4-dienoyl-CoA reductase from pRDR181, The PCR product was inserted into a pET15b expression vector and overexpressed in Escherichia coli. The soluble expressed protein can be separated into high- and low-activity fractions. The low-activity fraction can be converted to the high specific activity form by thermal annealing, suggesting it is a metastable misfolded form of the enzyme. Using ion-exchange and affinity chromatography, the enzyme has been purified to homogeneity and exhibits a single band on Coomassie blue-stained SDS-PAGE, The molecular mass of 32,413 Da determined by electrospray ionization-mass spectrometry indicates that the amino-terminal methionine had been removed. The Michaelis constants for trans-2, trans-4-hexadienoyl-CoA and NADPH were determined to be 0.46 and 2.5 mu M, respectively; a turnover number of 2.1 s(-1) was calculated, (C) 1999 Academic Press.
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页码:57 / 63
页数:7
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