Expression and purification of His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase wild-type and His352 mutant proteins.
Mitochondrial 3-ketoacyl-CoA thiolase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned a cDNA of rat mitochondrial 3-ketoacyl-CoA thiolase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned cDNA was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 92% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase was 25U/mg. It has been proposed that His352 is a catalytic residue responsible for activation of coenzyme A by deprotonation of a sulfhydryl group. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that His352 is a catalytic residue of rat mitochondrial 3-ketoacyl-CoA thiolase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial 3-ketoacyl-CoA thiolase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by 3-ketoacyl-CoA thiolase.
Zeng
Jia
J
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Li
Ding
D
D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D018076:DNA, Complementary; D006639:Histidine / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D006639:Histidine; D000102:Acetyl-CoA C-Acyltransferase