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

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.