Cloning, expression, and purification of the functional Δ3-Δ2-enoyl-CoA isomerase fusion protein

Delta(3)-Delta(2)-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the P-oxidation of unsaturated fatty acids. The cDNA of the full-length rat liver Delta(3)-Delta(2)-enoyl-CoA isomerase was previously cloned as pAG847. PCR methodologies were used to subclone the gene encoding the functional Delta(3)-Delta(2)-enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5' primer. The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli. The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity, column to apparent homogeneity based on Coomassie blue-stained SDS PAGE and the molecular weight of the protein Subunit was 30 kDa. The purified protein had a dimeric structure composed of identical Subunits, and the molecular v, eight of the enzyme determined by gel chromatography was 60 kDa. Kinetic studies have been carried out and K-M of 81 muM and V-max of 292 mumol/min/mg were determined. The specific activity of the protein is 201 U/mg. which is significantly higher than that reported before for the same protein isolated from a natural source. The one-step purification of the highly active Delta(3)-Delta(2)-enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues. (C) 2002 Elsevier Science (USA). All rights reserved.