Expression and assembly of a functional E1 component (α2β2) of mammalian branched-chain α-ketoacid dehydrogenase complex in Escherichia coli

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Abstract

We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain α-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1α and E1β subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1α subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1α fusion and E1β subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1α and E1β did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an α2β2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1α and E1β subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 α2β2 heterotetramer. By using this co- expression system, we also find that the E1α missense mutation (Tyr-393 → Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.

Original languageEnglish (US)
Pages (from-to)16601-16606
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number23
StatePublished - Jan 1 1992

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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