Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli.

摘要: Abstract We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature alpha and beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, was fused with ligand E. maltose-binding protein (MBP) through endoprotease Factor Xa-specific linker peptide. When co-expressed, MBP-E1 fusion subunits were shown to co-purify as that exhibited both activity binding competence for E2 component. In contrast, vitro mixing individually did not result assembly or produce activity. Following proteolytic removal peptide Xa, species eluted from Sephacryl S-300HR sizing column enzymatically 160-kDa species. The latter showed 1:1 stoichiometry, which consistent 2 structure. recovery this (estimated at 0.07% total lysate protein) low, majority lost insoluble aggregates. Our findings suggest concurrent same cellular compartment is important functional heterotetramer. By using co-expression system, we also find missense mutation (Tyr-393----Asn) characterized Mennonites maple syrup urine disease prevents soluble heterotetramers.