Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Expression of Human Dihydrolipoamide Dehydrogenase-Binding Protein in Excherichia coli

  • Lee, Jeong-Min (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University) ;
  • Ryou, Chong-Suk (Institute of Neurodegenerative Diseases, University of California San Francisco) ;
  • Kwon, Moo-Sik (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University)
  • Published : 2001.08.01
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Abstract

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

Keywords

References

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