Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli

We developed a bacterial expression system for the human alpha and betacDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putativemitochondrial matrix targeting presequences) were co-expressed inEscherichia coli on one plasmid vector with each cDNA having its ownIPTG-inducible promoter. Only negligible amounts of active PCC weremeasured despite the presence of both alpha and beta subunits as indicatedby Western blot analysis and the almost complete biotinylation of the alphasubunit. Co-expression of this plasmid with a second plasmid vectorover-expressing the E. coli chaperonin proteins, groES and groEL, resultedin a several hundred-fold increase in PCC specific activity, to a levelcomparable with that found in crude human liver extracts. PCC was partiallypurified on monomeric avidin affinity resin and the presence of both alphaand beta subunits was demonstrated, thereby confirming the assembly of bothsubunits into an active enzyme. Deficiency of either alpha PCC or beta PCCresults in propionic acidemia, an autosomal recessive disorder. We usedthis expression system to characterize one missense mutation previouslydescribed in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC.This mutation, when expressed in E.coli under the same conditions as thatof wild-type PCC, had null activity, despite the presence of assembledalpha PCC and beta PCC subunits. This bacterial expression system can beuseful for analysis of either alpha PCC or beta PCC mutations. Our findingsindicated that the groES and groEL chaperonin proteins were essential forfolding and assembly of the human PCC heteromeric subunits.